Methods of cancer treatment using activated T cells

ABSTRACT

Provided is a method of treating a cancer in an individual using activated T cells or PBMCs induced by antigen presenting cells (such as dendritic cells) loaded with a plurality of tumor antigen peptides. The method may further comprise administration of the antigen presenting cells loaded with the plurality of tumor antigen peptides to the individual. The methods may be used singly or in combination with an immune checkpoint inhibitor. Also provided are precision therapy methods customized for the individual using neoantigen peptides or based on the mutation load in the tumor of the individual, methods of preparing the activated T cells, methods of monitoring the treatment, methods of cloning tumor-specific T cell receptors, an isolated population of cells comprising the activated T cells, and compositions and kits useful for cancer immunotherapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/557,794, filed Sep. 12, 2017, which is a U.S. national phaseapplication of International Application No. PCT/CN2016/076165, filedMar. 11, 2016, which claims priority benefit of InternationalApplication No. PCT/CN2015/074227, filed Mar. 13, 2015, each of which isincorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 744852000110SEQLIST.TXT,date recorded: Mar. 10, 2021, size: 6 KB).

FIELD OF THE INVENTION

The present invention relates to the field of cancer immunotherapy. Morespecifically, this invention provides methods, compositions and kits fortreating cancer in an individual using activated T cells.

BACKGROUND OF THE INVENTION

The human body has an elaborate immune system to defend itself againstdiseases, including internal malignancies. Unleashing the body's ownimmune power to treat and prevent cancer has therefore been along-standing ideal in oncology. The natural immune response against atumor is typically elicited by tumor antigens, including mutatedproteins exclusively expressed in cancer cells, and tumor-associatedantigens (TAAs) overexpressed in cancer's tissue of origin but arenonetheless not completely recognized as “self”. Antigen presentingcells (APCs), notably dendritic cells (DCs), that encounter tumorantigens can process and present the tumor antigens on their cellsurface. Upon maturation, DCs loaded with tumor antigens can trigger Tcell response, which involves cytotoxic T cells, helper T cells, andfunctionally distinct effecter and memory T cells, against cancer cellshosting the tumor antigens. A particularly powerful type of T cellresponse involves production of cytotoxic T cells that can kill cancercells by releasing cytokines, enzymes, and cytotoxins, or by inducingpro-apoptosis signaling cascade via cell-cell interactions

Cancer immunotherapies aim to take advantage of the above process totreat cancer, but success has been rather limited until recently.Initial attempts have focused on developing cancer vaccines based onparticular antigen peptides, full-length antigen proteins, or viralvectors encoding tumor antigens. Few cancer vaccines have made into theclinics, and even fewer generated any impressive clinical outcome.Unlike traditional cancer therapy, such as chemotherapy, radiationtherapy and surgical resection, in general, the bodily response tocancer immunotherapy treatments, especially cancer vaccines, is muchdelayed because it takes time for APCs to process and present theantigen to T cells, and for T cells to mature and to elicit an immuneresponse. When a tumor is present in a patient, the cancer cells in thetumor already have mechanisms to escape surveillance by the immunesystem. Therefore, a successful tumor vaccine must be able to bypass thedefects in immune surveillance to elicit a strong immune response.Additionally, several bottleneck issues exist in cancer vaccines thatprevent the approach from producing specific and durable clinicaleffects. First, cancer cells, even of the same histological type, arerather heterogeneous in their genetic composition and expression profileamong different patients and among different lesions within the samepatient—a phenomenon well documented by a plethora of genetic data fromrecent next-generation sequencing experiments on cancer cells availablein the literature and public databases. Consequently, the limited numberof tumor antigen(s) in a particular cancer vaccine treatment is unlikelyto represent the spectrum of antigens characteristic of individualtumors in all patients. Secondly, many antigen moieties in cancervaccines are not effectively loaded onto the APCs due to serum half-lifeand bioavailability issues. Third, even when APCs are properly primed byantigens contained in cancer vaccines, lack of suitable activationsignals and microenvironments can result in production of the wrongsubpopulation of T cells, especially immunosuppressive regulatory Tcells (T_(REG)), which inhibit, instead of stimulate, immune responseagainst tumors. The origin of the last two issues has to do with thecomplete lack of control by clinicians in patients' actual response toany cancer vaccine once it is administered.

Cell-based cancer immunotherapy approach alleviates some of the abovechallenges in cancer vaccines by administering to patientsimmunity-mediating cells or cell products that are prepared underrelatively defined and controlled conditions. In particular, DC-basedmethods have garnered much interest, especially after the FDA approvedPROVENGE® (sipuleucel-T) in April 2010 for advanced prostate cancer. Atypical DC-based immunotherapy method involves isolating DCs from acancer patient, loading the DCs with a tumor antigen (or antigens,including tumor cell lysates and total mRNA) ex vivo, and thenadministering the DCs back to the patient to elicit cancer-killing Tcell response. PROVENGE®, for example, comprises exposing a patient'speripheral blood mononuclear cells (PBMCs) to a fusion proteincomprising a tumor-derived antigen coupled to a cytokine (such asGM-CSF), and then infusing the PBMCs (presumably containing activatedDCs that can present the tumor-derived antigen to T cells) to thepatient (see U.S. Pat. Nos. 5,976,546, 6,080,409, and 6,210,662). In thepivotal Phase III trial (Kantoff P W, Higano C S et al. (2010)“Sipuleucel-T immunotherapy for castration-resistant prostate cancer.” NJ Med 363:411-22), the specific embodiment of PROVENGE® was preparedusing a recombinant protein of prostatic acid phosphatase (PAP), aprostate cancer-associated antigen, fused to GM-CSF, a cytokine known toattract and induce DCs. Although PROVENGE® was able to prolong mediansurvival of the patients in the experimental group (25.8 months) ascompared to those in the control group (21.7 months), the clinical trialresults did not show evidence of statistically significant delay intumor progression or reduction in tumor size. More troubling is the factthat survival of individual patients does not seem to correlate withspecific T cell responses to either the fusion protein or PAP in thePROVENGE® treatment (Cheever M A, Higano C S (2011) “PROVENGE(Sipuleucel-T) in prostate cancer: the first FDA-approved therapeuticcancer vaccine.” Clin. Cancer Res. 17:3520-6).

A second method in the cell-based immunotherapy approach, named adoptivelymphocyte therapy, involves isolating tumor-infiltrating lymphocytes(TIL) from a patient's tumor, expanding the TILs ex vivo, and infusingthe TILs back to the patient after depleting the patient's nativenon-myeloid lymphocytes. Dramatic clinical responses, including completetumor recession and long disease-free survival, have been reported inclinical applications of adoptive lymphocyte therapy to patients withmelanoma (Restifo N P, Dudley M E, and Rosenberg S A. (2012) “Adoptiveimmunotherapy for cancer: harnessing the T cell response.” Nat. Rev.Immunol. 12: 269-81). It has further been shown that the clinicalbenefits of TIL are correlated with or resulting from tumor-specific Tcells present in the TIL population (Robbins P F et al. (2013) “Miningexomic sequencing data to identify mutated antigens recognized byadoptively transferred tumor-reactive T cells.” Nature Medicine 19:747-752; and Tran E et al. (2014) “Cancer immunotherapy based onmutation-specific CD4+ T cells in a patient with epithelial cancer”Science 344: 641-645). Recently, T cells with engineered T cellreceptors having modified affinity to certain tumor antigens or chimericantigen receptors (CAR-T) further expand the capacity of the adoptivelymphocyte therapy method by modifying the microenvironment of Tcell-tumor interactions. A major issue with the current adoptivelymphocytes therapy methods concerns multiple reports of severe adverseevents, including CNS toxicity, in clinical trials, likely having to dowith improper selection of targets (so called on-target off tumoreffect) and biased expansion of T cell populations. Another issue of theapproach is the lack of durable response in some patients, because ofrapidly developed immune tolerance to the tumor-specific antigenspresented on the infused T lymphocytes, as well as immune escape bycancer cells.

Immune tolerance and immune escape are often mediated by checkpointmolecules, or co-inhibitory signals, on cells interacting with T cellsin the microenvironment of the tumor site, in addition to an elevatedlevel of immunosuppressive cells, such as T_(REG) and MDSC(myeloid-derived suppressor cells). A well-studied pair of checkpointmolecules involves the immune-inhibitory PD-1 receptor on T cells andthe PD-L1 ligand on APCs (such as DCs), MDSCs and cancer cells. Bindingof PD-L1 to PD-1 triggers a signal to inhibit pro-inflammatory cytokine(e.g. IL-2) production and proliferation of cytotoxic T cells. In manyscenarios, PD-L1 binding to PD-1 triggers apoptosis of cytotoxic Tcells. On the other hand, the PD-1/PD-L1 signaling induces T_(REG)cells, which act to further inhibit T cells with tumor-attackingcapacity. Antibodies against PD-1, PD-L1, and other checkpoint molecules(such as CTLA-4 on T cells) are currently developed by severalpharmaceutical companies as a distinct approach in cancer immunotherapy,based on the theory that blockade of the T-cell checkpoints can helpovercome immune tolerance and immune escape in the tumor site. It isworth noting that the anti-tumor effects of the checkpoint blockadeapproach require pre-existence of tumor-specific T cells in vivo(Boussiotis V A (2014) “Somatic mutations and immunotherapy outcome withCTLA-4 blockade in melanoma” N. Engl. J. Med. 371:2230-2232; Wolchok J Dand Chan T A, (2014) “Cancer: antitumor immunity gets a boost” Nature515: 496-498).

Given the promises and challenges of the various cancer immunotherapyapproaches as described above, it is desirable to provide a new cancerimmunotherapy method that combines the advantages of the previousmethods while avoiding the known pitfalls.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods, compositions and kits fortreating cancer in an individual using activated T cells induced byantigen presenting cells (such as dendritic cells) loaded with aplurality of tumor antigen peptides.

One aspect of the present application provides a method of treating acancer in an individual (such as a human individual), comprisingadministering to the individual an effective amount of activated Tcells, wherein the activated T cells are prepared by co-culturing apopulation of T cells with a population of dendritic cells loaded with aplurality of tumor antigen peptides. In some embodiments, the individualhas previously been administered with an effective amount of dendriticcells loaded with the plurality of tumor antigen peptides. In someembodiments, the method further comprises administering to theindividual an effective amount of the dendritic cells loaded with theplurality of tumor antigen peptides. In some embodiments, the dendriticcells are administered prior (for example, about 7 days to about 14days, about 14 days to about 21 days, or about 7 days to about 21 daysprior) to the administration of the activated T cells.

In some embodiments according to any one of the methods described above,the method further comprises preparing the activated T cells byco-culturing the population of T cells with the population of dendriticcells loaded with the plurality of tumor antigen peptides prior to theadministration steps. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, or about 14 days to about 21days).

In some embodiments according to any one of the methods described above,the population of T cells is contacted with an immune checkpointinhibitor prior to the co-culturing. In some embodiments, the populationof T cells is co-cultured with the population of dendritic cells loadedwith the plurality of tumor antigen peptides in the presence of animmune checkpoint inhibitor. In some embodiments, the immune checkpointinhibitor is an inhibitor of an immune checkpoint molecule selected fromthe group consisting of PD-1, PD-L1, and CTLA-4.

In some embodiments according to any one of the methods described above,the method further comprises preparing the population of dendritic cellsloaded with the plurality of tumor antigen peptides. In someembodiments, the population of dendritic cells loaded with the pluralityof tumor antigen peptides is prepared by contacting a population ofdendritic cells with the plurality of tumor antigen peptides. In someembodiments, the population of dendritic cells loaded with the pluralityof tumor antigen peptides is prepared by contacting the population ofdendritic cells with the plurality of tumor antigen peptides in thepresence of a composition that facilitates the uptake of the pluralityof tumor antigen peptides by the dendritic cells.

In some embodiments according to any one of the methods described above,the population of T cells and the population of dendritic cells arederived from the same individual. In some embodiments, the population ofT cells and the population of dendritic cells are derived from theindividual being treated.

One aspect of the present application provides a method of preparing apopulation of activated T cells, the method comprising: (a) inducingdifferentiation of a population of monocytes into a population ofdendritic cells; (b) contacting the population of dendritic cells with aplurality of tumor antigen peptides to obtain a population of dendriticcells loaded with the plurality of tumor antigen peptides; and (c)co-culturing the population of dendritic cells loaded with the pluralityof tumor antigen peptides and a population of non-adherent PBMCs toobtain the population of activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs from an individual. In some embodiments, step b)comprises contacting the population of dendritic cells with theplurality of tumor antigen peptides in the presence of a compositionthat facilitates the uptake of the plurality of tumor antigen peptidesby the dendritic cells. In some embodiments, step b) further comprisescontacting the population of dendritic cells loaded with the pluralityof tumor antigen peptides with a plurality of Toll-like Receptor (TLR)agonists (such as polyIC, MALP, R848, or any combination thereof) toinduce maturation of the population of dendritic cells loaded with theplurality of tumor antigen peptides. In some embodiments, step c)further comprises contacting the population of activated T cells with aplurality of cytokines and optionally an anti-CD3 antibody to induceproliferation and differentiation of the population of activated Tcells. In some embodiments, the plurality of cytokines comprises IL-2,IL-7, IL-15 or IL-21. In some embodiments, the population ofnon-adherent PBMCs is contacted with an immune checkpoint inhibitorprior to the co-culturing. In some embodiments, step c) comprisesco-culturing the population of dendritic cells loaded with the pluralityof tumor antigen peptides and the population of non-adherent PBMCs inthe presence of an immune checkpoint inhibitor. In some embodiments, theimmune checkpoint inhibitor is an inhibitor of an immune checkpointmolecule selected from the group consisting of PD-1, PD-L1, and CTLA-4.

Further provided is a method of treating a cancer in an individual (suchas a human individual), comprising administering to the individual aneffective amount of a population of activated T cells prepared by themethod of any one of the methods described in the preceding paragraph.In some embodiments, the population of PBMCs is obtained from theindividual being treated.

In some embodiments according to any one of the methods of treating acancer as described above, the activated T cells are administered to theindividual for at least three times. In some embodiments, the intervalbetween each administration of the activated T cells is about 0.5 monthto about 5 months (such as about 0.5 month to about 2 month).

In some embodiments according to any one of the methods of treating acancer as described above, the activated T cells are administeredintravenously. In some embodiments, the activated T cells areadministered at a dose of at least about 3×10⁹ cells/individual. In someembodiments, the activated T cells are administered at about 1×10⁹ toabout 1×10¹⁰ cells/individual.

In some embodiments according to any one of the methods of treating acancer as described above, the dendritic cells loaded with the pluralityof tumor antigen peptides are administered for at least three times. Insome embodiments, the interval between each administration of thedendritic cells is about 0.5 month to about 5 months (such as about 0.5month to about 2 months).

In some embodiments according to any one of the methods of treating acancer as described above, the dendritic cells loaded with the pluralityof tumor antigen peptides are administered subcutaneously. In someembodiments, the dendritic cells are administered at a dose of about1×10⁶ to about 5×10⁶ cells/individual.

One aspect of the present application provides a method of treating acancer in an individual (such as a human individual), comprising: a)contacting a population of PBMCs with a plurality of tumor antigenpeptides to obtain a population of activated PBMCs, and b) administeringto the individual an effective amount of the activated PBMCs. In someembodiments, step (a) comprises contacting the population of PBMCs witha plurality of tumor antigen peptides in the presence of an immunecheckpoint inhibitor. In some embodiments, the immune checkpointinhibitor is an inhibitor of an immune checkpoint molecule selected fromthe group consisting of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA,and LAG-3. In some embodiments, the activated PBMCs are administered forat least three times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 0.5 month to about 5months (such as about 0.5 month to about 2 months). In some embodiments,the activated PBMCs are administered intravenously. In some embodiments,the activated PBMCs are administered at a dose of about 1×10⁹ to about1×10¹⁰ cells/individual.

In some embodiments according to any of the methods described above, theplurality of tumor antigen peptides is each about 20 to about 40 aminoacids long. In some embodiments, the plurality of tumor antigen peptidescomprises at least one peptide comprising an MHC-I epitope. In someembodiments, the at least one peptide comprising an MHC-I epitopefurther comprises additional amino acids flanking the epitope at theN-terminus, the C-terminus, or both.

In some embodiments according to any of the methods described above, theplurality of tumor antigen peptides comprises at least one peptidecomprising an MHC-II epitope. In some embodiments, the at least onepeptide comprising an MHC-II epitope further comprises additional aminoacids flanking the epitope at the N-terminus, the C-terminus, or both.

In some embodiments according to any of the methods described above, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides. In some embodiments, the plurality oftumor antigen peptides further comprises a second group of cancer-typespecific antigen peptides. In some embodiments, the first core groupcomprises about 10 to about 20 general tumor antigen peptides. In someembodiments, the second group comprises about 1 to about 10 cancer-typespecific antigen peptides.

In some embodiments according to any of the methods described above, theplurality of tumor antigen peptides comprises a neoantigen peptide. Insome embodiments, the neoantigen peptide is selected based on thegenetic profile of a tumor sample from the individual.

In some embodiments according to any of the methods of treating a canceras described above, the cancer is selected from the group consisting ofhepatic cellular carcinoma, cervical cancer, lung cancer, colorectalcancer, lymphoma, renal cancer, breast cancer, pancreatic cancer,gastric cancer, esophageal cancer, ovarian cancer, prostate cancer,nasopharyngeal cancer, melanoma and brain cancer.

In some embodiments according to any of the methods of treating a canceras described above, the method further comprises administering to theindividual an effective amount of an immune checkpoint inhibitor. Insome embodiments, the immune checkpoint inhibitor is an inhibitor of animmune checkpoint molecule selected from the group consisting of PD-1,PD-L1, and CTLA-4.

In some embodiments according to any of the methods of treating a canceras described above, the individual is selected for the method oftreating based on the mutation load in the cancer. In some embodiments,the individual has a low mutation load in the cancer. In someembodiments, the individual has a low mutation load in one or more MHCgenes. In some embodiments, the individual has no more than about 10mutations in the one or more MHC genes. In some embodiments, the one ormore MHC genes are MHC class I genes. In some embodiments, wherein theindividual is a human individual, the one or more MHC genes are selectedfrom the group consisting of HLA-A, HLA-B, HLA-C and B2M. In someembodiments, the individual has no mutation in B2M. In some embodiments,the individual has no mutation in the functional regions (such as leaderpeptide sequence, a1 domain, a2 domain, or a3 domain) of the one or moreMHC genes. In some embodiments, the mutation load of the cancer isdetermined by sequencing a tumor sample from the individual.

In some embodiments according to any of the methods of treating a canceras described above, the individual is selected for the method oftreating based on having one or more neoantigens in the cancer. In someembodiments, the individual has at least 5 neoantigens. In someembodiments, the method further comprises identifying a neoantigen ofthe cancer, and incorporating a neoantigen peptide in the plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen. In some embodiments, the neoantigen isidentified by sequencing a tumor sample from the individual. In someembodiments, said sequencing is targeted sequencing of cancer-associatedgenes. In some embodiments, the method further comprises determining theaffinity of the neoepitope to an MHC molecule. In some embodiments, themethod further comprises determining the affinity of the complexcomprising the neoepitope and an MHC molecule to a T cell receptor. Insome embodiments, the MHC molecule is an MHC class I molecule. In someembodiments, the MHC molecule is from the individual.

In some embodiments according to any of the methods of treating a canceras described above, the method further comprises monitoring theindividual after the administration of the activated T cells or theactivated PBMCs. In some embodiments, the monitoring comprisesdetermining the number of circulating tumor cells (CTC) in theindividual. In some embodiments, the monitoring comprises detecting aspecific immune response against the plurality of tumor antigen peptidesin the individual. In some embodiments, the plurality of tumor antigenpeptides is adjusted based on the specific immune response to provide aplurality of customized tumor antigen peptides. In some embodiments, themethod of treating is repeated using the plurality of customized tumorantigen peptides.

One aspect of the present application provides a method of cloning atumor-specific T cell receptor, comprising: (a) treating an individualwith any one of the methods of treating cancer as described above; (b)isolating a T cell from the individual, wherein the T cell specificallyrecognizes a tumor antigen peptide in the plurality of tumor antigenpeptides; and (c) cloning a T cell receptor from the T cell to providethe tumor-specific T cell receptor. In some embodiments, the individualhas a strong specific immune response against the tumor antigen peptide.In some embodiments, the T cell is isolated from a PBMC sample of theindividual. In some embodiments, the tumor antigen peptide is aneoantigen peptide.

Also provided are a tumor-specific T cell receptor cloned using any oneof the methods of cloning a tumor-specific T cell receptor as describedabove, an isolated T cell comprising the tumor-specific T cell receptor,and a method of treating a cancer in an individual comprisingadministering to the individual an effective amount of the isolated Tcell.

Further provided is an isolated population of cells (such as activated Tcells, or activated PBMCs) prepared by the method of any one of themethods of preparing as described above.

One aspect of the present application provides an isolated population ofcells comprising activated T cells, wherein less than about 1% of theactivated T cells are regulatory T (T_(REG)) cells.

In some embodiments according to any one of the isolated population ofcells described above, the isolated population of cells comprises about0.3% to about 0.5% CD4⁺CD25⁺Foxp3⁺ cells. In some embodiments, theisolated population of cells comprises about 65% to about 75% CD3⁺CD8⁺cells. In some embodiments, the isolated population of cells comprisesabout 16% to about 22% of CD3⁺CD4⁺ cells. In some embodiments, theisolated population of cells comprises about 13% to about 15% CD3⁺CD56⁺cells.

In some embodiments according to any one of the isolated population ofcells described above, the activated T cells are capable of elicitingspecific response to a plurality of tumor antigen peptides in vivo or exvivo. In some embodiments, the activated T cells express a plurality ofpro-inflammatory molecules. In some embodiments, the plurality ofpro-inflammatory molecules comprises IFNγ, TNFα, granzyme B, orperforin.

In some embodiments according to any one of the isolated population ofcells described above, the activated T cells have no or low expressionof a plurality of immunosuppressive cytokines. In some embodiments, theplurality of immunosuppressive cytokines comprises IL-10 or IL-4.

In some embodiments according to any one of the isolated population ofcells described above, less than about 5% of the activated T cellsexpress immune-inhibitory molecule PD-1.

In some embodiments according to any one of the isolated population ofcells described above, at least about 90% of the cells in the isolatedpopulation of cells are activated T cells.

One aspect of the present application provides a composition comprisingat least 10 tumor antigen peptides, wherein each of the at least 10tumor antigen peptides comprises at least one epitope selected from thegroup consisting of SEQ ID NOs: 1-35. In some embodiments, the at least10 tumor antigen peptides are selected from the group consisting of thetumor antigen peptides in FIG. 2C. In some embodiments, the at least 10tumor antigen peptides each comprises one or more epitopes encoded by acancer-associated gene selected from the group consisting of hTERT, p53,Survivin, N Y-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3,HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

Further provided are kits, medicines, and articles of manufacturecomprising any one of the compositions (such as isolated populations ofcells or compositions of tumor antigen peptides) as described above.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various embodiments described herein may be combinedto form other embodiments of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts two preferred embodiments of the MASCT method, includingtiming of the DC and T cell preparation steps, and administration(s) ofparticular cell-based compositions. Arrows below the time line indicateadministration steps.

FIGS. 2A-2C depict the cell manufacturing process of an exemplary MASCTmethod described in Example 1. FIG. 2A is a schematic diagramillustrating the cell manufacturing process of a preferred embodiment ofthe MASCT method. FIG. 2B shows an exemplary composition of HCC antigenpeptides pool loaded into DCs for the MASCT treatment in HCC patients.Some tumor antigen peptides have been used in clinical trials of cancerimmunotherapies; references to such DC vaccines, adoptive cell transfer(ACT) and peptides vaccines are included. OC: ovarian cancer; BC: breastcancer; PC: pancreatic cancer; LC: lung cancer; RCC: renal carcinoma;HCC: hepatocellular carcinoma. FIG. 2C shows a list of epitopescontained in the peptides pool of HCC.

FIG. 3 shows cellular uptake of tumor antigen peptides by iDCs. Humanmonocytes derived iDCs were pulsed with fluorescent labeled peptides ofsurvivin (second column on the left, 2.5 μg/ml) for 2 hours, followed bylabeling with DAPI (first column on the left) and LYSOTRACKER® (secondcolumn on the right) to identify nuclei and lysosomes, respectively.Fluorescent images were recorded with confocal microscopy (LeicaTCSST5), the scale bar is 7.5 μm, and the images are representative offour independent experiments.

FIGS. 4A-4B show characterization of mature DCs prepared in Example 1.FIG. 4A shows flow cytometry results of DCs before (gray peaks) andafter (black peaks) maturation with TLR agonists. Molecular markerstargeted by the antibodies used to separate cells in the flow cytometryexperiments are indicated above each chromatograph. Percentage of DCswith high expression levels (within the marked range) of each molecularmarker is indicated inside each panel. The results show that most of themature DCs exhibited a cell-surface expression signature to activatecytotoxic T cells. The DCs express MHC class II molecules andco-stimulatory signaling ligands CD86, CD80 and CD83, as well asmaturation receptor CCR7, but is low in expression level of CD14 that istypically expressed in immature DCs. FIG. 4B shows secretion level ofcytokines by the mature DCs prepared in Example 1. As expected offunctional, mature DCs, the DCs secreted high level of pro-inflammatorycytokine IL-12, but low level of immunosuppressive cytokine IL-10.

FIGS. 5A-5E show characterization of the activated T cells prepared inExample 1. FIG. 5A shows T cell expansion after 14 to 17 days culturingbased on cell counting using trypan blue exclusion. The median ofsamples from 10 patients is shown. FIG. 5B shows the percentages ofsubpopulations of T cells in the co-culture, indicating an extremely lowlevel of T_(REG) cells (CD4⁺CD25⁺Foxp3⁺, 0.4%±0.1%) among the activatedT cells. FIG. 5C shows pie charts displaying the percentages of T cellsubsets that co-expressed the cytokines (IFNγ and TNFα) and enzymegranzyme B. Mean±Standard Error of Measurement (SEM) of five patients isshown for each group. Triple producers: dark gray; double producers:light gray; single producers: black; non-producer: white. FIG. 5D shows3-dimensional flow cytometry chromatographs of activated T cellsprepared from patients' PBMC and co-cultured with pulsed DCs, which wasfurther stimulated with phorbol 12-myristate 13-acetate (PMA) for about4 hours. Data are representative of five independent experiments. Theactivated T cells contained large subpopulation of CD3⁺CD8⁺ cytotoxic Tcells, CD3⁺CD4⁺ helper T cells and CD3⁺CD56⁺ NK T cells, majority ofwhich had high intracellular production of pro-inflammatory cytokines(IFNγ and TNFα), and protease granzyme B. FIG. 5E shows 3-dimensionalflow cytometry chromatographs of non-activated T cells isolated frompatients stimulated with PMA for about 4 hours. The non-activated Tcells had only low expression levels of IFNγ, TNFα and granzyme B.

FIGS. 6A-6F depict molecular and functional characterizations of theactivated T cells prepared in Example 1. FIG. 6A shows secretion ofvarious cytokines by the activated T cells. The resulting cellsgenerated from HCC patients secreted significant amount of IFNγ andTNFα, but little to no IL10 and IL4. Mean±SEM is shown of 6 patients.FIGS. 6B-6C show reduced expression frequency of PD-1 on the surface ofCD3⁺CD8⁺ (FIG. 6B) and CD3⁺CD4⁺ (FIG. 6C) subsets of T cells isolatedfrom HCC patients compared to health donors. The expression percentageand statistic of 7 patients are shown. FIG. 6D shows reduction of thefrequency of PD-1 expressing T cells in the CD3⁺CD8⁺ subsets of T cellsafter ex vivo activation. FIG. 6E shows reduction of the frequency ofPD-1 expressing T cells in the CD3⁺CD4⁺ subsets of T cells after ex vivoactivation. The expression percentage and statistic of 7 patients areshown. FIG. 6F shows HLA (or MEW) restricted cytotoxicity of theactivated T cells. The activated T cells generated from PBMCs of HLA-A2⁺patients (n=7, left group) exhibited greater levels of cytotoxicactivity to the HCC cell line HepG2 (white bars, HLA-A2⁺) than to HuH-7cells (hashed bars, HLA-A2), while activated T cells generated fromPBMCs of HLA-A2⁻ patients (n=7, right group) exhibited similar levels ofcytotoxicity to these two cell lines. The relative ratio of effector Tcells (activated T cells prepared) to target cells (HepG2 or HuH-7cells; E:T ratio) in each cell lysis experiment was about 40:1.

FIG. 7A depicts a flow chart illustrating inclusion and exclusion ofpatients in the retrospective analysis of clinical data of a MASCTtreatment as described in Example 1.

FIG. 7B depicts a schematic diagram of the retrospective analysis ofstage B (according to Barcelona Clinic Liver Cancer stagingclassification) HCC patients continuously treated and regularlyfollowed-up.

FIG. 8A shows characteristics, treatment and RECIST evaluation ofpatients with hepatocellular carcinoma (B stage) in the control groupanalyzed in Example 1.

FIG. 8B shows characteristics, treatments, and RECIST evaluation ofpatients with hepatocellular carcinoma (B stage) who received onlyconventional therapy during 1 year after diagnosis (Group Con, n=17).

FIG. 9A shows characteristics, treatment and RECIST evaluation ofpatients with hepatocellular carcinoma (B stage) in the MASCT treatmentgroup analyzed in Example 1.

FIG. 9B shows characteristics, treatments and RECIST evaluation ofpatients with hepatocellular carcinoma (B stage) who received multipletreatments of MASCT during 1 year after diagnosis (Group Con+MASCT,n=15).

FIG. 10A shows a summary of comparison of patients between the controlgroup and the MASCT treatment group analyzed in Example 1.

FIG. 10B shows characteristics of patients with hepatocellular carcinoma(B stage) enrolled in the retrospective analysis.

FIGS. 11A-11F depict immune responses raised in patients with HCC afterMASCT treatment(s) as described in Example 1. FIG. 11A shows significantdecrease in the percentage of T_(REG) in PBMCs of 4 patients after theyreceived 3 MASCT treatments. The expression percentage and statistics of4 patients are shown. FIG. 11B shows increase in percentage ofproliferating T cells in PBMC samples from 7 different HCC patients whoreceived MASCT treatments. FIG. 11C shows increase in percentage ofINFγ-producing cytotoxic T cells (CD8+INFγ+) in PBMC samples from 7different HCC patients who received MASCT treatments. FIG. 11D showsflow cytometry chromatographs of a PBMC sample from an HCC patient whoreceived MASCT treatments. The results indicate that the INFγ-producingcytotoxic T cells (CD8+INFγ+) co-expressed CD27 and CD28, suggesting ahigh potential to acquire an immune memory of the HCC-specific T cellresponse. FIG. 11E shows an increase in intracellular production of IFNγby CD8⁺ T cells from patients with HCC after 3 MASCT treatments. PBMCswere isolated from patient before and after 3 MASCT treatmentsrespectively to measure T cell response. FIG. 11F shows specificproliferation of T cells in that sequentially increased in patientsduring multiple treatments of MASCT cell therapy. PBMCs were isolatedfrom patients before and after 1 and 3 MASCT treatment(s) respectively.T cell proliferations of 2 patients were measured by EdU(5-ethynyl-2′-deoxyuridine) staining. In FIG. 11B-11F, the specific Tcell responses were measured after stimulating the PBMCs with the HCCantigen peptides pool (HCC-pep). Control responses were measured afterstimulating the PBMCs with a pool of irrelevant peptides (ir-pep,control). All fold changes are calculated by normalizing the specificresponse value to the control response value.

FIGS. 12A-12D show specific immune responses against HCC antigenpeptides in patients in Example 1. Average specific immune responsesagainst individual HCC antigen peptides in HCC patients after multipleMASCT treatments (FIG. 12A; n=6) and in HCC patients without any MASCTtreatment (FIG. 12B; n=5). FIG. 12C shows specific immune responseagainst each kind of HCC antigen peptides in one patient before (emptybar) and after 3 MASCT treatments (hashed bar). FIG. 12D showssequential increase in specific immune response against each kind of HCCantigen peptides in a second HCC patient during multiple MASCTtreatments (white bar: before treatment; gray: after 1 treatment;hashed: after 3 treatments). The IFNγ secretion of patient's PBMCsstimulated with individual HCC antigen peptides was calculated byELISPOT. The results were shown in the mean±SEM fold change of IFNγsecretion compared to non-stimulated PBMCs. The numbers indicated theresponding patients/total patients. The higher dashed line indicated acut-off value of 1.5 fold increase. W/O: without stimulation.

FIGS. 13A-13F show clinical data of Patient WJ with metastatic cervicalcancer treated with 7 MASCT treatments. FIG. 13A, FIG. 13B, FIG. 13C,and FIG. 13D are ECT results of the patent taken in December 2013 (priorto any MASCT treatments), in June 2014 (after 10 local radiotherapytreatments followed by 3 MASCT treatments), and in December 2014 (aftera total of 7 MASCT treatments). The arrows and circles point to themetastasis site on the right sacroiliac joint bone, showing reduction ofthe metastatic tumor and no additional metastasis in response to MASCTtreatments. FIGS. 13E and 13F show specific immune response against thecervical carcinoma antigen peptide pool (CC pep pool), and each type ofantigen peptides in the pool after MASCT treatments. PBMCs were isolatedfrom the patient before any MASCT treatment and after a total of 6 MASCTtreatments, and were stimulated with the CC pep pool and each individualantigen peptides within the pool. Each column represents the level ofimmune response of the patient's PBMC after MASCT treatments againsteach antigen peptide (or CC pep pool) as measured by fold changes ofIFNγ (Y-axis) with respect to the corresponding response of thepatient's PBMC prior to MASCT treatments. W/O=response withoutstimulation with any antigen peptide. ENV refers to experiment withirrelevant peptide. The dotted line indicates a threshold of no elevatedimmune response as measured by IFNγ fold changes. Arrows point tospecific antigen peptides that elicited elevated immune response asmeasured by IFNγ fold changes.

FIG. 14 shows a summary of the patient's treatment history in Example 2.

FIG. 15 shows a schematic of exemplary experimental setups for preparingactivated T cells.

FIG. 16A shows FACS results of mature dendritic cells using anti-PD-L1antibody and anti-CD11c antibody. FIG. 16B shows PD-1 expression levelsof T cells in the PBMC samples from four different donors before andafter 8 days of activation.

FIG. 17A shows percentage of peptide-specific CD8⁺ T cells in co-culturesamples with 1 time or 2 times of antigen peptide stimulation, with orwithout the presence of anti-PD-1 antibody (nivolumab). FIG. 17B showspercentage of functional peptide-specific CD8⁺ T cells in co-culturesamples with 1 time or 2 times of antigen peptide stimulation, with orwithout the presence of anti-PD-1 antibody. FIG. 17C shows percentage ofpeptide-specific CD8⁺ T cells in co-culture samples with 1 time or 2times of antigen peptide stimulation, with or without the presence ofanti-PD-1 antibody (SHR-1210 or nivolumab). FIG. 17D shows percentage offunctional peptide-specific CD8⁺ T cells in co-culture samples with 1time or 2 times of antigen peptide stimulation, with or without thepresence of anti-PD-1 antibody (SHR-1210 or nivolumab).

FIG. 18 shows a schematic of exemplary experimental setups for preparingactivated T cells.

FIG. 19A shows percentage of peptide-specific CD8⁺ T cells in co-culturesamples with 1 time of antigen peptide stimulation and cultured for 5days or 10 days, with or without the presence of anti-PD-1 antibody(SHR-1210 or nivolumab). FIG. 19B shows percentage of peptide-specificCD8⁺ T cells in co-culture samples with 1 time of antigen peptidestimulation and cultured for 10 days or 2 times of antigen peptidestimulation and cultured for 5 days after the second stimulation, withor without the presence of anti-PD-1 antibody (SHR-1210 or nivolumab).FIG. 19C shows percentage of functional peptide-specific CD8⁺ T cells inco-culture samples with 1 time of antigen peptide stimulation andcultured for 10 days or 2 times of antigen peptide stimulation andcultured for 5 days after the second stimulation, with or without thepresence of anti-PD-1 antibody (SHR-1210 or nivolumab).

FIGS. 20A-20B show the total T cell counts in the co-cultures from PBMCsof two different donors over time with or without the presence ofanti-PD-1 antibody (SHR-1210 or nivolumab).

FIGS. 21A-21B show the percentage of cells expressing PD-1 on the cellsurface in the co-cultures from PBMCs of two different donors over timewith or without the presence of anti-PD-1 antibody (SHR-1210 ornivolumab).

FIG. 22 shows statistical data of Next Generation Sequencing (NGS) of333 cancer-associated genes in tumor samples and clinical evaluations ofthe 5 patients of Example 5.

FIGS. 23A-23B depict the DMM classification analysis of 35 tumorsamples. FIG. 23A depicts the best fit classification group number. FIG.23B depicts the DMM classification plot of 35 tumor samples. 14 sampleswere clustered into DMM 1 group (red, in box A), and 21 samples wereclustered into DMM 0 group (green, in box B).

FIGS. 24A-24B depict clustering analysis of 35 tumor tissue samplesbased on mutation load of the 333 oncogenes in each sample. FIG. 24Ashows a heatmap of 35 tumor samples clustered based on the mutation loaddetected in each of the 333 cancer-associated genes, with cancerclinical type, MMR deficiency type (0: MMR deficient, 1:MMR proficient)and DMM groups labeled. FIG. 24B shows a bar chart of HLA-I genemutation load of each samples, ordered with the same order matching thatin FIG. 24A. The black line marks 6 mutations in the HLA-I mutationload.

FIGS. 25A-25B depict statistical analysis of HLA-I gene mutation load ofeach tumor tissue sample within the two DMM groups.

FIGS. 26A-26E depict the CT scans of patient 3-HJL at 5 time points. CTscans in FIG. 26A show sarcoidosis in both lobes of the lung, with thebiggest one having diameter of 2 cm.

FIG. 26B shows similar sarcoidosis after 2 cycles of chemotherapy. FIG.26C shows no improvement on the lung sarcoidosis after 4 cycles ofchemotherapy. CT scans in FIG. 26D depict the shrinkage of the lungsarcoidosis of ˜50% in size after 3 cycles of combined therapy of PD-1inhibitor (KEYTRUDA®) and MASCT. FIG. 26E shows the disappearance ofsarcoidosis from both lobes of the lung after 5 cycles of combinedtherapy of PD-1 inhibitor (KEYTRUDA®) and MASCT.

FIGS. 27A-27D depict CT scans of patient 4-LKS at 4 time points. CTscans in FIG. 27A indicate brain metastasis, with the tumor size of ˜3cm. FIG. 27B shows tumor shrinkage after radiation therapy.Re-examination of CT scans in FIG. 27C indicate tumor shrinkage andalleviated brain edema. FIG. 27D shows further alleviation on tumor andedema status.

FIG. 28 shows an overview flow chart of an exemplary precision MASCTusing neoantigen peptides predicted based on sequencing results of apatient's tumor sample, and prognosis based on HLA mutation status.

FIG. 29A shows candidate neoantigens of a patient based on sequencinganalysis of the patient's tumor sample. FIG. 29B shows continuousmonitoring results of circulating tumor cells (CTC) in the patientbefore and after MASCT treatments. FIG. 29C shows ELISPOT results ofPBMC from the patient challenged with various antigen peptides after thepatient received three cycles of precision MASCT treatments.

FIG. 30A shows clinical characteristics of 45 patients withhepatocellular carcinoma (HCC) who received MASCT treatments.

FIG. 30B shows results of routine blood examination of the 45 patientsbefore and after MASCT treatments.

FIG. 30C shows liver and renal function parameters of the 45 patientsbefore and after MASCT treatments.

FIG. 30D shows ALT and AST levels in 8 HCC patients before MASCTtreatments and during the course of 5 MASCT treatments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses novel cell-based immunotherapy methods,collectively referred to as Multiple Antigen Specific Cell Therapy(MASCT), which are useful for treating a variety of cancers, as well asdelaying the progression of, preventing relapse or metastasis of, and/oralleviating a symptom of a cancer in an individual. The methods in someembodiments utilize activated T cells induced by dendritic cells (DCs)loaded with a plurality of tumor antigen peptides. The T cells and DCs,for example, can be derived from the individual's own peripheral bloodmononuclear cells (PBMCs). Multiple-antigen loaded DCs can be preparedby exposure of DCs (such as immature DCs) to a plurality of tumorantigen peptides comprising general tumor antigen peptides, andoptionally cancer-type specific antigen peptides. Activated T cells canbe prepared by co-culturing a population of T cells with themultiple-antigen loaded DCs. Optionally, the population of T cells iscontacted with an immune checkpoint inhibitor prior to and/or during theco-culturing. The activated T cells are administered to the individual,which can elicit an adoptive immune response against the tumor antigensin vivo. Optionally, the multiple-antigen loaded DCs can be administeredto the individual to trigger active immunity against the tumor antigens.Alternatively, PBMC-based MASCT methods comprising administration ofactivated PBMCs are provided. Any of the MASCT methods described hereinmay be used singly or in combination with an immune checkpoint inhibitor(such as PD-1 inhibitor) for treating cancer in the individual.

The present invention further provides precision MASCT treatment methodstailored to the individual being treated, such as the genetic profile ofthe tumor of the individual. For example, the individual can be selectedfor the MASCT treatment based on the mutation load (such as in one ormore MHC genes) in the tumor of the individual. The individual may alsobe selected for the MASCT treatment based on the number of neoantigensfound in the tumor of the individual. In some cases, one or moreneoantigens can be identified by sequencing a tumor sample from theindividual. Neoantigen peptides may be designed based on the neoantigensof the individual, and incorporated in the plurality of tumor antigenpeptides in order to provide a precision MASCT to the individual. Insome embodiments, the individual is monitored for specific immuneresponse against each tumor antigen peptide after a MASCT treatmentcycle to allow customization of the plurality of tumor antigen peptidesbased on the strength of the specific immune response for future MASCTtreatment cycles. Additionally, tumor-specific T cell receptors (TCR),which specifically recognize an epitope in a tumor antigen peptide andelicit a strong specific immune response, can be cloned from theindividual after the MASCT, and used for further precision immunotherapyon the individual.

The MASCT (including PBMC-based MASCT and precision MASCT) methods andcompositions provided herein can alleviate many of the technical issuesencountered by the previous cancer immunotherapy methods discussed inthe background section. For example, by exposing DCs to a pool of tumorantigen peptides in vitro, a multitude of tumor antigens, as opposed toa single tumor antigen in many cancer vaccines or in PROVEGENE®, arepresented by the DCs, allowing a wider spectrum of response againsttumors of different antigen expression profiles within the sameindividual or in different individuals, as long as the tumors share oneor more specific tumor antigens in the pool. The tumor antigen peptidespool can further be customized according to specific conditions of eachindividual, such as cancer type, viral infection status, and response toindividual antigen peptides, to achieve optimal therapeutic effects ineach treatment. Unlike cancer vaccines and DC-based therapies, the MASCTtreatment methods comprise administering activated T cells, bypassingthe in vivo T cell induction step of previous immunotherapies, which isnormally associated with a weakened response in cancer patients owing tothe various immune defects caused by tumor cells; thereby, the MASCTmethod may elicit strong, rapid and specific T cell response againstcancer cells. Furthermore, the activated T cells have very low T_(REG)level and PD-1 expression, leading to reduced immunosuppression oncancer-attacking T cells, thereby delaying cancer immune escape. Takentogether, the present invention provides an effective, durable, andwidely applicable cancer immunotherapy method to satisfy the tremendousunmet medical needs of cancer patients, especially when currentstandard-of-care treatments fail or are unavailable.

Definitions

Terms are used herein as generally used in the art, unless otherwisedefined as follows.

As used herein, “treatment” or “treating” is an approach for obtainingbeneficial or desired results including clinical results. For purposesof this invention, beneficial or desired clinical results include, butare not limited to, one or more of the following: decreasing one moresymptoms resulting from the disease, diminishing the extent of thedisease, stabilizing the disease (e.g., preventing or delaying theworsening of the disease), preventing or delaying the spread (e.g.,metastasis) of the disease, preventing or delaying the occurrence orrecurrence of the disease, delay or slowing the progression of thedisease, ameliorating the disease state, providing a remission (whetherpartial or total) of the disease, decreasing the dose of one or moreother medications required to treat the disease, delaying theprogression of the disease, increasing the quality of life, and/orprolonging survival. Also encompassed by “treatment” is a reduction ofpathological consequence of cancer. The methods of the inventioncontemplate any one or more of these aspects of treatment.

The term “individual” or “patient” is used synonymously herein todescribe a mammal, including humans. An individual includes, but is notlimited to, human, bovine, horse, feline, canine, rodent, or primate. Insome embodiments, the individual is human. In some embodiments, anindividual suffers from a disease, such as cancer. In some embodiments,the individual is in need of treatment.

As used herein, “delaying” the development of cancer means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease. This delay can be of varying lengths of time, depending on thehistory of the disease and/or individual being treated. As is evident toone skilled in the art, a sufficient or significant delay can, ineffect, encompass prevention, in that the individual does not developthe disease. A method that “delays” development of cancer is a methodthat reduces probability of disease development in a given time frameand/or reduces the extent of the disease in a given time frame, whencompared to not using the method. Such comparisons are typically basedon clinical studies, using a statistically significant number ofindividuals. Cancer development can be detectable using standardmethods, including, but not limited to, computerized axial tomography(CAT Scan), Magnetic Resonance Imaging (MRI), abdominal ultrasound,clotting tests, arteriography, or biopsy. Development may also refer tocancer progression that may be initially undetectable and includesoccurrence, recurrence, and onset.

As is understood in the art, an “effective amount” refers to an amountof a composition (e.g. multiple-antigen loaded DCs, activated T cells,activated PMBCs, or isolated T cells), first therapy, second therapy, ora combination therapy sufficient to produce a desired therapeuticoutcome (e.g., reducing the severity or duration of, stabilizing theseverity of, or eliminating one or more symptoms of cancer). Fortherapeutic use, beneficial or desired results include, e.g., decreasingone or more symptoms resulting from the disease (biochemical, histologicand/or behavioral), including its complications and intermediatepathological phenotypes presented during development of the disease,increasing the quality of life of those suffering from the disease,decreasing the dose of other medications required to treat the disease,enhancing effect of another medication, delaying the progression of thedisease, and/or prolonging survival of patients.

The methods may be practiced in an adjuvant setting. “Adjuvant setting”refers to a clinical setting in which an individual has had a history ofa proliferative disease, particularly cancer, and generally (but notnecessarily) been responsive to therapy, which includes, but is notlimited to, surgery (such as surgical resection), radiotherapy, andchemotherapy. However, because of their history of the proliferativedisease (such as cancer), these individuals are considered at risk ofdevelopment of the disease. Treatment or administration in the “adjuvantsetting” refers to a subsequent mode of treatment. The degree of risk(i.e., when an individual in the adjuvant setting is considered as “highrisk” or “low risk”) depends upon several factors, most usually theextent of disease when first treated.

The methods provided herein may also be practiced in a “neoadjuvantsetting,” i.e., the method may be carried out before theprimary/definitive therapy. In some embodiments, the individual haspreviously been treated. In some embodiments, the individual has notpreviously been treated. In some embodiments, the treatment is a firstline therapy.

As used herein, by “combination therapy” is meant that a first agent beadministered in conjunction with another agent. “In conjunction with”refers to administration of one treatment modality in addition toanother treatment modality, such as administration of activated T cellsor PBMCs described herein in addition to administration of another agent(such as an immune checkpoint inhibitor) to the same individual. Assuch, “in conjunction with” refers to administration of one treatmentmodality before, during, or after delivery of the other treatmentmodality to the individual. Such combinations are considered to be partof a single treatment regimen or regime.

The term “simultaneous administration,” as used herein, means that afirst therapy and second therapy in a combination therapy areadministered with a time separation of no more than about 15 minutes,such as no more than about any of 10, 5, or 1 minutes. When the firstand second therapies are administered simultaneously, the first andsecond therapies may be contained in the same composition (e.g., acomposition comprising both a first and second therapy) or in separatecompositions (e.g., a first therapy in one composition and a secondtherapy is contained in another composition).

As used herein, the term “sequential administration” means that thefirst therapy and second therapy in a combination therapy areadministered with a time separation of more than about 15 minutes, suchas more than about any of 20, 30, 40, 50, 60, or more minutes. Eitherthe first therapy or the second therapy may be administered first. Thefirst and second therapies are contained in separate compositions, whichmay be contained in the same or different packages or kits.

As used herein, the term “concurrent administration” means that theadministration of the first therapy and that of a second therapy in acombination therapy overlap with each other.

As used herein, by “pharmaceutically acceptable” or “pharmacologicallycompatible” is meant a material that is not biologically or otherwiseundesirable, e.g., the material may be incorporated into apharmaceutical composition administered to an individual without causingany significant undesirable biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Pharmaceutically acceptable carriers orexcipients have preferably met the required standards of toxicologicaland manufacturing testing and/or are included on the Inactive IngredientGuide prepared by the U.S. Food and Drug administration.

An “adverse event” or “AE” as used herein refers to any untoward medicaloccurrence in an individual receiving a marketed pharmaceutical productor in an individual who is participating on a clinical trial who isreceiving an investigational or non-investigational pharmaceuticalagent. The AE does not necessarily have a causal relationship with theindividual's treatment. Therefore, an AE can be any unfavorable andunintended sign, symptom, or disease temporally associated with the useof a medicinal product, whether or not considered to be related to themedicinal product. An AE includes, but is not limited to: anexacerbation of a pre-existing illness; an increase in frequency orintensity of a pre-existing episodic event or condition; a conditiondetected or diagnosed after study drug administration even though it mayhave been present prior to the start of the study; and continuouslypersistent disease or symptoms that were present at baseline and worsenfollowing the start of the study. An AE generally does not include:medical or surgical procedures (e.g., surgery, endoscopy, toothextraction, or transfusion); however, the condition that leads to theprocedure is an adverse event; pre-existing diseases, conditions, orlaboratory abnormalities present or detected at the start of the studythat do not worsen; hospitalizations or procedures that are done forelective purposes not related to an untoward medical occurrence (e.g.,hospitalizations for cosmetic or elective surgery or social/convenienceadmissions); the disease being studied or signs/symptoms associated withthe disease unless more severe than expected for the individual'scondition; and overdose of study drug without any clinical signs orsymptoms.

A “serious adverse event” or (SAE) as used herein refers to any untowardmedical occurrence at any dose including, but not limited to, that: a)is fatal; b) is life-threatening (defined as an immediate risk of deathfrom the event as it occurred); c) results in persistent or significantdisability or incapacity; d) requires in-patient hospitalization orprolongs an existing hospitalization (exception: Hospitalization forelective treatment of a pre-existing condition that did not worsenduring the study is not considered an adverse event. Complications thatoccur during hospitalization are AEs and if a complication prolongshospitalization, then the event is serious); e) is a congenitalanomaly/birth defect in the offspring of an individual who receivedmedication; or f) conditions not included in the above definitions thatmay jeopardize the individual or may require intervention to prevent oneof the outcomes listed above unless clearly related to the individual'sunderlying disease. “Lack of efficacy” (progressive disease) is notconsidered an AE or SAE. The signs and symptoms or clinical sequelaeresulting from lack of efficacy should be reported if they fulfill theAE or SAE definitions.

The following definitions may be used to evaluate response based ontarget lesions: “complete response” or “CR” refers to disappearance ofall target lesions; “partial response” or “PR” refers to at least a 30%decrease in the sum of the longest diameters (SLD) of target lesions,taking as reference the baseline SLD; “stable disease” or “SD” refers toneither sufficient shrinkage of target lesions to qualify for PR, norsufficient increase to qualify for PD, taking as reference the nadir SLDsince the treatment started; and “progressive disease” or “PD” refers toat least a 20% increase in the SLD of target lesions, taking asreference the nadir SLD recorded since the treatment started, or, thepresence of one or more new lesions.

The following definitions of response assessments may be used toevaluate a non-target lesion: “complete response” or “CR” refers todisappearance of all non-target lesions; “stable disease” or “SD” refersto the persistence of one or more non-target lesions not qualifying forCR or PD; and “progressive disease” or “PD” refers to the “unequivocalprogression” of existing non-target lesion(s) or appearance of one ormore new lesion(s) is considered progressive disease (if PD for theindividual is to be assessed for a time point based solely on theprogression of non-target lesion(s), then additional criteria arerequired to be fulfilled.

“Progression free survival” (PFS) indicates the length of time duringand after treatment that the cancer does not grow. Progression-freesurvival includes the amount of time individuals have experienced acomplete response or a partial response, as well as the amount of timeindividuals have experienced stable disease.

“Predicting” or “prediction” is used herein to refer to the likelihoodthat an individual is likely to respond either favorably or unfavorablyto a treatment regimen.

As used herein, “at the time of starting treatment” or “baseline” refersto the time period at or prior to the first exposure to the treatment.

As used herein, “sample” refers to a composition which contains amolecule which is to be characterized and/or identified, for example,based on physical, biochemical, chemical, physiological, and/or geneticcharacteristics.

“Cells,” as used herein, is understood to refer not only to theparticular individual cell, but to the progeny or potential progeny ofsuch a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

The term “peptide” refers to a polymer of amino acids no more than about100 amino acids (including fragments of a protein), which may be linearor branched, comprise modified amino acids, and/or be interrupted bynon-amino acids. The term also encompasses an amino acid polymer thathas been modified naturally or by intervention, including, for example,disulfide bond formation, glycosylation, lipidation, acetylation,phosphorylation, or any other manipulation or modification. Alsoincluded within this term are, for example, polypeptides containing oneor more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Thepeptides described herein may be naturally-occurring, i.e., obtained orderived from a natural source (e.g., blood) or synthesized (e.g.,chemically synthesized or by synthesized by recombinant DNA techniques).

As used herein, “a plurality of tumor antigen peptides,” “multiple tumorantigen peptides,” “a pool of tumor antigen peptides” and “a tumorantigen peptides pool” are used interchangeably to refer to acombination of more than one tumor antigen peptides.

As used herein, “dendritic cells loaded with a plurality of tumorantigen peptides” and “multiple-antigen loaded dendritic cells” are usedinterchangeably to refer to dendritic cells that have enhancedpresentation of more than one tumor antigen peptides among the pluralityof tumor antigen peptides. Likewise, “APCs loaded with a plurality oftumor antigen peptides” are used interchangeably with “multiple-antigenloaded APCs” to refer to antigen processing cells that have enhancedpresentation of more than one tumor antigen peptides among the pluralityof tumor antigen peptides.

As used herein, “activated T cells” refer to a population of monoclonal(e.g. encoding the same TCR) or polyclonal (e.g. with clones encodingdifferent TCRs) T cells that have T cell receptors that recognize atleast one tumor antigen peptide. Activated T cells may contain one ormore subtypes of T cells, including, but not limited to, cytotoxic Tcells, helper T cells, natural killer T cells, γδ T cells, regulatory Tcells, and memory T cells.

As used herein, “immune checkpoint inhibitor” refers to a molecule or anagent (including an antibody) that inhibits or blocks an inhibitoryimmune checkpoint molecule on an immune cell (such as T cell, or PBMC)or a tumor cell. “Immune checkpoint molecules” include molecules thatturn up an immune signal (i.e., “co-stimulatory molecules”), ormolecules that turn down an immune signal (i.e., “inhibitory immunecheckpoint molecules”) against a tumor cell.

As used herein, “mutation load” refers to the total number of mutationsaccumulated at one or more loci (such as gene) in the genome of a cell(such as a tumor cell). The mutations include, but are not limited to,point mutation, insertion, deletion, frame shift mutation, gene fusion,and copy number variation. The mutations may or may not adversely affectthe physical/chemical properties, and/or functions of the productencoded by the locus.

As used herein, “T cell receptor” or “TCR” refers to an endogenous orengineered T cell receptor comprising an extracellular antigen bindingdomain that binds to a specific antigen epitope bound in an MHCmolecule. A TCR may comprise a TCRα polypeptide chain and a TCR βpolypeptide chain. “Tumor-specific TCR” refers to a TCR thatspecifically recognizes a tumor antigen expressed by a tumor cell.

As used herein, the term “HLA” or “Human Leukocyte Antigen” refers tothe human genes that encode for the MHC (Major HistocompatibilityComplex) proteins on the surface of cells that are responsible forregulation of the immune system. “HLA-I” or “HLA class I” refers tohuman MHC class I genes, including HLA-A, HLA-B, HLA-C, HLA-E, HLA-F,HLA-G, and β2-microglobulin loci. “HLA-II” or “HLA class II” refers tohuman MHC class II genes, including HLA-DPA1, HLA-DPB1, HLA-DQA1,HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM,HLA-DOA, and HLA-DOB loci.

The term “antibody” used herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), multispecific antibodies (e.g., bispecificantibodies), and antibody fragments so long as they exhibit the desiredbiological activity.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. In someembodiments, the antibody fragment described herein is anantigen-binding fragment. Examples of antibody fragments include Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

As use herein, the term “specifically binds to,” “recognizes,”“specifically recognizes,” “targets,” or is “specific for” refers tomeasurable and reproducible interactions such as binding between atarget and an antibody, or a receptor and a ligand, or a receptor and anepitope/MHC complex, which is determinative of the presence of thetarget in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody that binds toor specifically binds to a target (which can be an epitope) is anantibody that binds this target with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other targets. Inone embodiment, the extent of binding of an antibody to an unrelatedtarget is less than about 10% of the binding of the antibody to thetarget as measured, e.g., by a radioimmunoassay (RIA). In certainembodiments, an antibody that specifically binds to an antigen peptide(or an epitope) has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10nM, ≤1 nM, or ≤0.1 nM. In certain embodiments, an antibody specificallybinds to an epitope on a protein that is conserved among the proteinfrom different species. In another embodiment, specific binding caninclude, but does not require exclusive binding.

It is understood that aspect and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

The term “about X-Y” used herein has the same meaning as “about X toabout Y.”

As used herein, reference to “not” a value or parameter generally meansand describes “other than” a value or parameter. For example, the methodis not used to treat cancer of type X means the method is used to treatcancer of types other than X.

As used herein and in the appended claims, the singular forms “a,” “or,”and “the” include plural referents unless the context clearly dictatesotherwise.

MASCT Method

The present invention provides cell-based immunotherapy methods oftreating cancer in an individual, collectively referred to as MultipleAntigen Specific Cell Therapy (MASCT). The methods make use of antigenpresenting cells (APCs, such as dendritic cells) loaded with a pluralityof tumor antigen peptides, and activated T cells induced by themultiple-antigen loaded APCs. Both the multiple-antigen loaded APCs andthe activated T cells are capable of eliciting tumor antigen-specific Tcell response in vivo and ex vivo, including response by cytotoxic Tcells and helper T cells, as well as generating an immune memory throughmemory T cells. Therefore, in various embodiments of the MASCT method,multiple-antigen loaded APCs (such as dendritic cells), activated Tcells, co-culture of APCs and T cells (including activated PBMCs), orany combination thereof can be administered to an individual to treat acancer or neoplastic condition, or to prevent tumor relapse, progressionor metastasis.

The present invention in one aspect provides a method of treating acancer in an individual, comprising administering to the individual aneffective amount of activated T cells, wherein the activated T cells areprepared by co-culturing a population of T cells with a population ofantigen presenting cells (such as dendritic cells) loaded with aplurality of tumor antigen peptides. In some embodiments, the activatedT cells are administered intravenously. In some embodiments, theactivated T cells are administered for at least three times. In someembodiments, the activated T cells and the population of antigenpresenting cells are from the same individual. In some embodiments, theactivated T cells and/or the population of antigen presenting cells arefrom the individual being treated. In some embodiments, the populationof antigen presenting cells is a population of dendritic cells, B cells,or macrophages. In some embodiments, the antigen presenting cells aredendritic cells.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing a population of T cells with a population of antigenpresenting cells (such as dendritic cells) loaded with a plurality oftumor antigen peptides, and wherein the individual has previously beenadministered an effective amount of antigen presenting cells loaded withthe plurality of tumor antigen peptides. In some embodiments, theinterval between administration of the antigen presenting cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, or about 14 days to about 21days). In some embodiments, the antigen presenting cells areadministered subcutaneously. In some embodiments, the antigen presentingcells are administered for at least three times. In some embodiments,the activated T cells are administered intravenously. In someembodiments, the activated T cells are administered for at least threetimes. In some embodiments, the activated T cells and the population ofantigen presenting cells are from the same individual. In someembodiments, the activated T cells and/or the population of antigenpresenting cells are from the individual being treated. In someembodiments, the population of antigen presenting cells is a populationof dendritic cells, B cells, or macrophages. In some embodiments, theantigen presenting cells are dendritic cells.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) administering to the individual aneffective amount of antigen presenting cells (such as dendritic cells)loaded with the plurality of tumor antigen peptides; and (b)administering to the individual an effective amount of activated Tcells, wherein the activated T cells are prepared by co-culturing apopulation of T cells with a population of antigen presenting cellsloaded with a plurality of tumor antigen peptides. In some embodiments,the antigen presenting cells are administered about 7 days to about 21days (such as about 7 days to about 14 days, or about 14 days to about21 days) prior to the administration of the activated T cells. In someembodiments, the antigen presenting cells are administered for at leastthree times. In some embodiments, the activated T cells are administeredintravenously. In some embodiments, the activated T cells areadministered for at least three times. In some embodiments, theactivated T cells and the population of antigen presenting cells arefrom the same individual. In some embodiments, the activated T cellsand/or the population of antigen presenting cells are from theindividual being treated. In some embodiments, the population of antigenpresenting cells is a population of dendritic cells, B cells, ormacrophages. In some embodiments, the antigen presenting cells aredendritic cells.

Any suitable antigen presenting cells may be used in the MASCT methods,including, but not limited to, dendritic cells, B cells, andmacrophages. In some embodiments, the antigen presenting cells aredendritic cells.

Thus, in some embodiments, there is provided a method of treating acancer in an individual, comprising administering to the individual aneffective amount of activated T cells, wherein the activated T cells areprepared by co-culturing a population of T cells with a population ofdendritic cells loaded with a plurality of tumor antigen peptides. Insome embodiments, the activated T cells are prepared by co-culturing apopulation of T cells with the population of dendritic cells loaded withthe plurality of tumor antigen peptides prior to the administration. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, theactivated T cells are administered intravenously. In some embodiments,the activated T cells are administered for at least three times. In someembodiments, the activated T cells and the population of dendritic cellsare from the same individual. In some embodiments, the activated T cellsand/or the population of dendritic cells are from the individual beingtreated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing a population of T cells with a population of dendriticcells loaded with a plurality of tumor antigen peptides, and wherein theindividual has previously been administered an effective amount ofdendritic cells loaded with the plurality of tumor antigen peptides. Insome embodiments, the dendritic cells are administered about 7 days toabout 21 days (such as about 7 days to about 14 days, or about 14 daysto about 21 days) prior to the administration of the activated T cells.In some embodiments, the dendritic cells loaded with the plurality oftumor antigen peptides are administered subcutaneously. In someembodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the activated T cells and thepopulation of dendritic cells are from the same individual. In someembodiments, the activated T cells and/or the population of dendriticcells are from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) administering to the individual aneffective amount of dendritic cells loaded with a plurality of tumorantigen peptides; and (b) administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing a population of T cells with a population of dendriticcells loaded with the plurality of tumor antigen peptides. In someembodiments, the dendritic cells are administered about 7 days to about21 days (such as about 7 days to about 14 days, or about 14 days toabout 21 days) prior to the administration of the activated T cells. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, theactivated T cells are administered intravenously. In some embodiments,the activated T cells are administered for at least three times. In someembodiments, the activated T cells and the population of dendritic cellsare from the same individual. In some embodiments, the activated T cellsand/or the population of dendritic cells are from the individual beingtreated.

In addition to the administration step(s), some embodiments of the MASCTmethod further comprise one or two of the following cell preparationsteps: 1) preparation of the population of antigen presenting cells(such as dendritic cells) loaded with the plurality of tumor antigenpeptides; and 2) preparation of the activated T cells. In someembodiments, the activated T cells are prepared by co-culturing apopulation of T cells with the population of antigen presenting cellsloaded with the plurality of tumor antigen peptides prior to theadministration. In some embodiments, the population of T cells isco-cultured with the population of antigen presenting cells loaded withthe plurality of tumor antigen peptides for about 7 days to about 21days (such as about 7 days to about 14 days, about 14 days to about 21days, about 10 days, about 14 days, or about 21 days). In someembodiments, the population of antigen presenting cells loaded with theplurality of tumor antigen peptides is prepared by contacting apopulation of antigen presenting cells with the plurality of tumorantigen peptides. In some embodiments, the population of antigenpresenting cells is contacted with the plurality of tumor antigenpeptides in the presence of a composition that facilitates the uptake ofthe plurality of tumor antigen peptides by the antigen presenting cells.In some embodiments, the population of T cells is contacted with animmune checkpoint inhibitor prior to the co-culturing. In someembodiments, the population of T cells is co-cultured with thepopulation of antigen presenting cells in the presence of an immunecheckpoint inhibitor. In some embodiments, the population of T cells andthe population of antigen presenting cells are derived from the sameindividual. In some embodiments, the population of T cells and thepopulation of antigen presenting cells are derived from the individualbeing treated.

Thus, in some embodiments, there is provided a method of treating acancer in an individual, comprising: (a) co-culturing a population ofdendritic cells loaded with a plurality of tumor antigen peptides and apopulation of T cells to obtain a population of activated T cells; and(b) administering to the individual an effective amount of the activatedT cells. In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days, about 14 days, or about 21 days). In some embodiments,the population of T cells is derived from the non-adherent portion of apopulation of peripheral blood mononuclear cells (PBMCs). In someembodiments, the co-culturing further comprises contacting the activatedT cells with a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21,or any combination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of T cells is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4)prior to and/or during the co-culturing. In some embodiments, thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides is prepared by contacting a population of dendritic cells withthe plurality of tumor antigen peptides. In some embodiments, thepopulation of T cells and the population of dendritic cells are derivedfrom the same individual. In some embodiments, the population of Tcells, the population of dendritic cells, the population of PBMCs, orany combination thereof is derived from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) co-culturing a population of dendriticcells loaded with a plurality of tumor antigen peptides and a populationof T cells to obtain a population of activated T cells; and (b)administering to the individual an effective amount of the activated Tcells, wherein the individual has previously been administered aneffective amount of dendritic cells loaded with the plurality of tumorantigen peptides. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,or about 10 days). In some embodiments, the population of T cells isderived from the non-adherent portion of a population of peripheralblood mononuclear cells (PBMCs). In some embodiments, the co-culturingfurther comprises contacting the activated T cells with a plurality ofcytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof)and optionally an anti-CD3 antibody. In some embodiments, the populationof T cells is contacted with an immune checkpoint inhibitor (such as aninhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or during theco-culturing. In some embodiments, the population of dendritic cellsloaded with the plurality of tumor antigen peptides is prepared bycontacting a population of dendritic cells with the plurality of tumorantigen peptides. In some embodiments, the population of T cells and thepopulation of dendritic cells are derived from the same individual. Insome embodiments, the population of T cells, the population of dendriticcells, the population of PBMCs, or any combination thereof is derivedfrom the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) administering to the individual aneffective amount of dendritic cells loaded with a plurality of tumorantigen peptides; (b) co-culturing a population of dendritic cellsloaded with the plurality of tumor antigen peptides and a population ofT cells to obtain a population of activated T cells; and (c)administering to the individual an effective amount of the activated Tcells. In some embodiments, the interval between the administration ofthe dendritic cells and the administration of the activated T cells isabout 7 days to about 21 days (such as about 7 days to about 14 days,about 14 days to about 21 days, about 10 days or about 14 days). In someembodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, theactivated T cells are administered intravenously. In some embodiments,the activated T cells are administered for at least three times. In someembodiments, the population of T cells is co-cultured with thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides for about 7 days to about 21 days (such as about 7 days toabout 10 days, about 10 days to about 15 days, about 15 days to about 21days, about 14 days to about 21 days, or about 10 days). In someembodiments, the population of T cells is derived from the non-adherentportion of a population of peripheral blood mononuclear cells (PBMCs).In some embodiments, the co-culturing further comprises contacting theactivated T cells with a plurality of cytokines (such as IL-2, IL-7,IL-15, IL-21, or any combination thereof) and optionally an anti-CD3antibody. In some embodiments, the population of T cells is contactedwith an immune checkpoint inhibitor (such as an inhibitor of PD-1,PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of dendritic cells loaded with the pluralityof tumor antigen peptides is prepared by contacting a population ofdendritic cells with the plurality of tumor antigen peptides. In someembodiments, the population of T cells and the population of dendriticcells are derived from the same individual. In some embodiments, thepopulation of T cells, the population of dendritic cells, the populationof PBMCs, or any combination thereof is derived from the individualbeing treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) preparing a population of dendritic cellsloaded with a plurality of tumor antigen peptides; (b) co-culturing thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of T cells to obtain a population of activatedT cells; and (c) administering to the individual an effective amount ofthe activated T cells. In some embodiments, the dendritic cells loadedwith the plurality of tumor antigen peptides are administeredsubcutaneously. In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered for at least threetimes. In some embodiments, the activated T cells are administeredintravenously. In some embodiments, the activated T cells areadministered for at least three times. In some embodiments, thepopulation of T cells is co-cultured with the population of dendriticcells loaded with the plurality of tumor antigen peptides for about 7days to about 21 days (such as about 7 days to about 14 days, about 14days to about 21 days, or about 10 days). In some embodiments, thepopulation of T cells is derived from the non-adherent portion of apopulation of peripheral blood mononuclear cells (PBMCs). In someembodiments, the co-culturing further comprises contacting the activatedT cells with a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21,or any combination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of T cells is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4)prior to and/or during the co-culturing. In some embodiments, thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides is prepared by contacting a population of dendritic cells withthe plurality of tumor antigen peptides. In some embodiments, thepopulation of T cells and the population of dendritic cells are derivedfrom the same individual. In some embodiments, the population of Tcells, the population of dendritic cells, the population of PBMCs, orany combination thereof is derived from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) preparing a population of dendritic cellsloaded with a plurality of tumor antigen peptides; (b) co-culturing thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of T cells to obtain a population of activatedT cells; and (c) administering to the individual an effective amount ofthe activated T cells, wherein the individual has previously beenadministered an effective amount of dendritic cells loaded with theplurality of tumor antigen peptides. In some embodiments, the intervalbetween the administration of the dendritic cells and the administrationof the activated T cells is about 7 days to about 21 days (such as about7 days to about 14 days, about 14 days to about 21 days, about 10 daysor about 14 days). In some embodiments, the dendritic cells loaded withthe plurality of tumor antigen peptides are administered subcutaneously.In some embodiments, the dendritic cells loaded with the plurality oftumor antigen peptides are administered for at least three times. Insome embodiments, the activated T cells are administered intravenously.In some embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,or about 10 days). In some embodiments, the population of T cells isderived from the non-adherent portion of a population of peripheralblood mononuclear cells (PBMCs). In some embodiments, the co-culturingfurther comprises contacting the activated T cells with a plurality ofcytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof)and optionally an anti-CD3 antibody. In some embodiments, the populationof T cells is contacted with an immune checkpoint inhibitor (such as aninhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or during theco-culturing. In some embodiments, the population of dendritic cellsloaded with the plurality of tumor antigen peptides is prepared bycontacting a population of dendritic cells with the plurality of tumorantigen peptides. In some embodiments, the population of T cells and thepopulation of dendritic cells are derived from the same individual. Insome embodiments, the population of T cells, the population of dendriticcells, the population of PBMCs, or any combination thereof is derivedfrom the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) preparing a population of dendritic cellsloaded with a plurality of tumor antigen peptides; (b) administering tothe individual an effective amount of the dendritic cells loaded withthe plurality of tumor antigen peptides; (c) co-culturing the populationof dendritic cells loaded with the plurality of tumor antigen peptidesand a population of T cells to obtain a population of activated T cells;and (d) administering to the individual an effective amount of theactivated T cells. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,or about 10 days). In some embodiments, the population of T cells isderived from the non-adherent portion of a population of peripheralblood mononuclear cells (PBMCs). In some embodiments, the co-culturingfurther comprises contacting the activated T cells with a plurality ofcytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof)and optionally an anti-CD3 antibody. In some embodiments, the populationof T cells is contacted with an immune checkpoint inhibitor (such as aninhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or during theco-culturing. In some embodiments, the population of T cells and thepopulation of dendritic cells are derived from the same individual. Insome embodiments, the population of dendritic cells loaded with theplurality of tumor antigen peptides is prepared by contacting apopulation of dendritic cells with the plurality of tumor antigenpeptides. In some embodiments, the population of T cells and thepopulation of dendritic cells are derived from the same individual. Insome embodiments, the population of T cells, the population of dendriticcells, the population of PBMCs, or any combination thereof is derivedfrom the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells, wherein the population of monocytes and the population ofnon-adherent PBMCs are obtained from a population of PBMCs; and (d)administering to the individual an effective amount of the activated Tcells. In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,or about 10 days). In some embodiments, the co-culturing furthercomprises contacting the activated T cells with a plurality of cytokines(such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) andoptionally an anti-CD3 antibody. In some embodiments, the population ofnon-adherent PBMCs is contacted with an immune checkpoint inhibitor(such as an inhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or duringthe co-culturing. In some embodiments, the population of PBMCs isderived from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells; and (d) administering to the individual an effective amount ofthe activated T cells, wherein the population of monocytes and thepopulation of non-adherent PBMCs are obtained from a population ofPBMCs, and wherein the individual has previously been administered aneffective amount of dendritic cells loaded with the plurality of tumorantigen peptides. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the co-culturing is for about 7 daysto about 21 days (such as about 7 days to about 14 days, about 14 daysto about 21 days, or about 10 days). In some embodiments, theco-culturing further comprises contacting the activated T cells with aplurality of cytokines (such as IL-2, IL-7, IL-15, IL-21, or anycombination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, orCTLA-4) prior to and/or during the co-culturing. In some embodiments,the population of PBMCs and/or dendritic cells is obtained from theindividual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) administering to the individual an effectiveamount of the dendritic cells loaded with the plurality of tumor antigenpeptides; (d) co-culturing the population of dendritic cells loaded withthe plurality of tumor antigen peptides and a population of non-adherentPBMCs to obtain the population of activated T cells; and (e)administering to the individual an effective amount of the activated Tcells, wherein the population of monocytes and the population ofnon-adherent PBMCs are obtained from a population of PBMCs. In someembodiments, the interval between the administration of the dendriticcells and the administration of the activated T cells is about 7 days toabout 21 days (such as about 7 days to about 14 days, about 14 days toabout 21 days, about 10 days or about 14 days). In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, or about 10 days). Insome embodiments, the co-culturing further comprises contacting theactivated T cells with a plurality of cytokines (such as IL-2, IL-7,IL-15, IL-21, or any combination thereof) and optionally an anti-CD3antibody. In some embodiments, the population of non-adherent PBMCs iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of PBMCs is obtained from the individualbeing treated.

The methods described herein are suitable for treating various cancers,such as cancers described herein, including a cancer selected from thegroup consisting of hepatocellular carcinoma, cervical cancer, lungcancer, colorectal cancer, lymphoma, renal carcinoma, breast cancer,pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer,prostate cancer, nasopharyngeal carcinoma, melanoma, and brain cancer.The methods are applicable to cancers of all stages, including earlystage, advanced stage and metastatic cancer. In some embodiments, thecancer is solid tumor. In some embodiments, the cancer is liquid cancer.

In some embodiments, the method reduces the severity of one or moresymptoms associated with the cancer by at least about any of 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% compared to thecorresponding symptom in the same individual prior to treatment orcompared to the corresponding symptom in other individuals not receivingthe treatment method. In some embodiments, the method delays progressionof the cancer.

Examples of cancers that may be treated by the methods described hereininclude, but are not limited to, adenocortical carcinoma, agnogenicmyeloid metaplasia, anal cancer, appendix cancer, astrocytoma (e.g.,cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g.,extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignantfibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma,cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuseastrocytoma, anaplastic (malignant) astrocytoma), malignant glioma,ependymoma, oligodenglioma, meningioma, craniopharyngioma,haemangioblastomas, medulloblastoma, supratentorial primitiveneuroectodermal tumors, visual pathway and hypothalamic glioma, andglioblastoma), breast cancer, bronchial adenomas/carcinoids, carcinoidtumor (e.g., gastrointestinal carcinoid tumor), carcinoma of unknownprimary, central nervous system lymphoma, cervical cancer, colon cancer,colorectal cancer, chronic myeloproliferative disorders, endometrialcancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing'sfamily of tumors, eye cancer (e.g., intraocular melanoma andretinoblastoma), gallbladder cancer, gastric (stomach) cancer,gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST),germ cell tumor, (e.g., extracranial, extragonadal, ovarian),gestational trophoblastic tumor, head and neck cancer, hepatocellular(liver) cancer (e.g., hepatic carcinoma and heptoma), hypopharyngealcancer, islet cell carcinoma (endocrine pancreas), laryngeal cancer,laryngeal cancer, leukemia (except for T-cell leukemia), lip and oralcavity cancer, oral cancer, liver cancer, lung cancer (e.g., small celllung cancer, non-small cell lung cancer, adenocarcinoma of the lung, andsquamous carcinoma of the lung), lymphoma (except for T-cell lymphoma),medulloblastoma, melanoma, mesothelioma, metastatic squamous neckcancer, mouth cancer, multiple endocrine neoplasia syndrome,myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases,nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma,neuroblastoma, neuroendocrine cancer, oropharyngeal cancer, ovariancancer (e.g., ovarian epithelial cancer, ovarian germ cell tumor,ovarian low malignant potential tumor), pancreatic cancer, parathyroidcancer, penile cancer, cancer of the peritoneal, pharyngeal cancer,pheochromocytoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, pleuropulmonary blastoma,primary central nervous system lymphoma (microglioma), pulmonarylymphangiomyomatosis, rectal cancer, renal carcinoma, renal pelvis andureter cancer (transitional cell cancer), rhabdomyosarcoma, salivarygland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cellcarcinoma), melanoma, and Merkel cell carcinoma), small intestinecancer, squamous cell cancer, testicular cancer, throat cancer, thyroidcancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvarcancer, Wilms' tumor, abnormal vascular proliferation associated withphakomatoses, edema (such as that associated with brain tumors), andMeigs' syndrome.

Thus, in some embodiments, there is provided a method of treatinghepatocellular carcinoma (HCC) in an individual comprising administeringto the individual an effective amount of activated T cells, wherein theactivated T cells are prepared by co-culturing a population of T cellswith a population of antigen presenting cells (such as dendritic cells)loaded with a plurality of tumor antigen peptides. In some embodiments,the individual has previously been administered with an effective amountof antigen presenting cells loaded with the plurality of tumor antigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of antigenpresenting cells loaded with the plurality of tumor antigen peptidesprior to the administration of the activated T cells. In someembodiments, the HCC is early stage HCC, non-metastatic HCC, primaryHCC, advanced HCC, locally advanced HCC, metastatic HCC, HCC inremission, or recurrent HCC. In some embodiments, the HCC is localizedresectable (i.e., tumors that are confined to a portion of the liverthat allows for complete surgical removal), localized unresectable(i.e., the localized tumors may be unresectable because crucial bloodvessel structures are involved or because the liver is impaired), orunresectable (i.e., the tumors involve all lobes of the liver and/or hasspread to involve other organs (e.g., lung, lymph nodes, bone). In someembodiments, the HCC is, according to TNM classifications, a stage Itumor (single tumor without vascular invasion), a stage II tumor (singletumor with vascular invasion, or multiple tumors, none greater than 5cm), a stage III tumor (multiple tumors, any greater than 5 cm, ortumors involving major branch of portal or hepatic veins), a stage IVtumor (tumors with direct invasion of adjacent organs other than thegallbladder, or perforation of visceral peritoneum), N1 tumor (regionallymph node metastasis), or M1 tumor (distant metastasis). In someembodiments, the HCC is, according to AJCC (American Joint Commission onCancer) staging criteria, stage T1, T2, T3, or T4 HCC. In someembodiments, the HCC is any one of liver cell carcinomas, fibrolamellarvariants of HCC, and mixed hepatocellularcholangiocarcinomas. In someembodiments, the HCC is caused by Hepatitis B Virus (HBV) infection.

In some embodiments, there is provided a method of treating lung cancerin an individual comprising administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing (such as in the presence of an immune checkpointinhibitor) a population of T cells with a population of antigenpresenting cells (such as dendritic cells) loaded with a plurality oftumor antigen peptides. In some embodiments, the individual haspreviously been administered with an effective amount of antigenpresenting cells loaded with the plurality of tumor antigen peptides. Insome embodiments, the method further comprises administering to theindividual an effective amount of antigen presenting cells loaded withthe plurality of tumor antigen peptides prior to the administration ofthe activated T cells. In some embodiments, the lung cancer is anon-small cell lung cancer (NSCLC). Examples of NSCLC include, but arenot limited to, large-cell carcinoma (e.g., large-cell neuroendocrinecarcinoma, combined large-cell neuroendocrine carcinoma, basaloidcarcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, andlarge-cell carcinoma with rhabdoid phenotype), adenocarcinoma (e.g.,acinar, papillary (e.g., bronchioloalveolar carcinoma, nonmucinous,mucinous, mixed mucinous and nonmucinous and indeterminate cell type),solid adenocarcinoma with mucin, adenocarcinoma with mixed subtypes,well-differentiated fetal adenocarcinoma, mucinous (colloid)adenocarcinoma, mucinous cystadenocarcinoma, signet ring adenocarcinoma,and clear cell adenocarcinoma), neuroendocrine lung tumors, and squamouscell carcinoma (e.g., papillary, clear cell, small cell, and basaloid).In some embodiments, the NSCLC may be, according to TNM classifications,a stage T tumor (primary tumor), a stage N tumor (regional lymph nodes),or a stage M tumor (distant metastasis).

In some embodiments, the lung cancer is a carcinoid (typical oratypical), adenosquamous carcinoma, cylindroma, or carcinoma of thesalivary gland (e.g., adenoid cystic carcinoma or mucoepidermoidcarcinoma). In some embodiments, the lung cancer is a carcinoma withpleomorphic, sarcomatoid, or sarcomatous elements (e.g., carcinomas withspindle and/or giant cells, spindle cell carcinoma, giant cellcarcinoma, carcinosarcoma, or pulmonary blastoma). In some embodiments,the lung cancer is small cell lung cancer (SCLC; also called oat cellcarcinoma). The small cell lung cancer may be limited-stage, extensivestage or recurrent small cell lung cancer. In some embodiments, theindividual may be a human who has a gene, genetic mutation, orpolymorphism suspected or shown to be associated with lung cancer (e.g.,SASH1, LATS1, IGF2R, PARK2, KRAS, PTEN, Kras2, Krag, Pas1, ERCC1, XPD,IL8RA, EGFR, α₁-AD, EPHX, MMP1, MMP2, MMP3, MMP12, IL1β, RAS, and/orAKT) or has one or more extra copies of a gene associated with lungcancer.

In some embodiments, there is provided a method of treating cervicalcancer in an individual comprising administering to the individual aneffective amount of activated T cells, wherein the activated T cells areprepared by co-culturing (such as in the presence of an immunecheckpoint inhibitor) a population of T cells with a population ofantigen presenting cells (such as dendritic cells) loaded with aplurality of tumor antigen peptides. In some embodiments, the individualhas previously been administered with an effective amount of antigenpresenting cells loaded with the plurality of tumor antigen peptides. Insome embodiments, the method further comprises administering to theindividual an effective amount of antigen presenting cells loaded withthe plurality of tumor antigen peptides prior to the administration ofthe activated T cells. In some embodiments, the cervical cancer is earlystage cervical cancer, non-metastatic cervical cancer, locally advancedcervical cancer, metastatic cervical cancer, cervical cancer inremission, unresectable cervical cancer, cervical cancer in an adjuvantsetting, or cervical cancer in a neoadjuvant setting. In someembodiments, the cervical cancer is caused by human papillomavirus (HPV)infection. In some embodiments, the cervical cancer may be, according toTNM classifications, a stage T tumor (primary tumor), a stage N tumor(regional lymph nodes), or a stage M tumor (distant metastasis). In someembodiments, the cervical cancer is any of stage 0, stage I (Tis, N0,M0), stage IA (T1a, N0, M0), stage D3 (T1b, N0, M0), stage IIA (T2a, N0,M0), stage IIB (T2b, N0, M0), stage IIIA (T3a, N0, M0), stage IIIB (T3b,N0, M0, or T1-3, N1, M0) stage IVA (T4, N0, M0), or stage IVB (T1-T3,N0-N1, M1) cervical cancer. In some embodiments, the cervical cancer iscervical squamous cell carcinoma, cervical adenocarcinoma, oradenosquamous carcinoma.

In some embodiments, there is provided a method of treating breastcancer in an individual comprising administering to the individual aneffective amount of activated T cells, wherein the activated T cells areprepared by co-culturing (such as in the presence of an immunecheckpoint inhibitor) a population of T cells with a population ofantigen presenting cells (such as dendritic cells) loaded with aplurality of tumor antigen peptides. In some embodiments, the individualhas previously been administered with an effective amount of antigenpresenting cells loaded with the plurality of tumor antigen peptides. Insome embodiments, the method further comprises administering to theindividual an effective amount of antigen presenting cells loaded withthe plurality of tumor antigen peptides prior to the administration ofthe activated T cells. In some embodiments, the breast cancer is earlystage breast cancer, non-metastatic breast cancer, locally advancedbreast cancer, metastatic breast cancer, hormone receptor positivemetastatic breast cancer, breast cancer in remission, breast cancer inan adjuvant setting, ductal carcinoma in situ (DCIS), invasive ductalcarcinoma (IDC), or breast cancer in a neoadjuvant setting. In someembodiments, the breast cancer is hormone receptor positive metastaticbreast cancer. In some embodiments, the breast cancer (which may be HER2positive or HER2 negative) is advanced breast cancer. In someembodiments, the breast cancer is ductal carcinoma in situ. In someembodiments, the individual may be a human who has a gene, geneticmutation, or polymorphism associated with breast cancer (e.g., BRCA1,BRCA2, ATM, CHEK2, RAD51, AR, DIRAS3, ERBB2, TP53, AKT, PTEN, and/orPI3K) or has one or more extra copies of a gene (e.g., one or more extracopies of the HER2 gene) associated with breast cancer.

In some embodiments, there is provided a method of treating pancreaticcancer in an individual comprising administering to the individual aneffective amount of activated T cells, wherein the activated T cells areprepared by co-culturing (such as in the presence of an immunecheckpoint inhibitor) a population of T cells with a population ofantigen presenting cells (such as dendritic cells) loaded with aplurality of tumor antigen peptides. In some embodiments, the individualhas previously been administered with an effective amount of antigenpresenting cells loaded with the plurality of tumor antigen peptides. Insome embodiments, the method further comprises administering to theindividual an effective amount of antigen presenting cells loaded withthe plurality of tumor antigen peptides prior to the administration ofthe activated T cells. In some embodiments, the pancreatic cancerincludes, but is not limited to, serous microcystic adenoma, intraductalpapillary mucinous neoplasm, mucinous cystic neoplasm, solidpseudopapillary neoplasm, pancreatic adenocarcinoma, pancreatic ductalcarcinoma, or pancreatoblastoma. In some embodiments, the pancreaticcancer is any of early stage pancreatic cancer, non-metastaticpancreatic cancer, primary pancreatic cancer, resected pancreaticcancer, advanced pancreatic cancer, locally advanced pancreatic cancer,metastatic pancreatic cancer, unresectable pancreatic cancer, pancreaticcancer in remission, recurrent pancreatic cancer, pancreatic cancer inan adjuvant setting, or pancreatic cancer in a neoadjuvant setting.

In some embodiments, there is provided a method of treating ovariancancer in an individual comprising administering to the individual aneffective amount of activated T cells, wherein the activated T cells areprepared by co-culturing (such as in the presence of an immunecheckpoint inhibitor) a population of T cells with a population ofantigen presenting cells (such as dendritic cells) loaded with aplurality of tumor antigen peptides. In some embodiments, the individualhas previously been administered with an effective amount of antigenpresenting cells loaded with the plurality of tumor antigen peptides. Insome embodiments, the method further comprises administering to theindividual an effective amount of antigen presenting cells loaded withthe plurality of tumor antigen peptides prior to the administration ofthe activated T cells. In some embodiments, the ovarian cancer isovarian epithelial cancer. Exemplary ovarian epithelial cancerhistological classifications include: serous cystomas (e.g., serousbenign cystadenomas, serous cystadenomas with proliferating activity ofthe epithelial cells and nuclear abnormalities but with no infiltrativedestructive growth, or serous cystadenocarcinomas), mucinous cystomas(e.g., mucinous benign cystadenomas, mucinous cystadenomas withproliferating activity of the epithelial cells and nuclear abnormalitiesbut with no infiltrative destructive growth, or mucinouscystadenocarcinomas), endometrioid tumors (e.g., endometrioid benigncysts, endometrioid tumors with proliferating activity of the epithelialcells and nuclear abnormalities but with no infiltrative destructivegrowth, or endometrioid adenocarcinomas), clear cell (mesonephroid)tumors (e.g., benign clear cell tumors, clear cell tumors withproliferating activity of the epithelial cells and nuclear abnormalitiesbut with no infiltrative destructive growth, or clear cellcystadenocarcinomas), unclassified tumors that cannot be allotted to oneof the above groups, or other malignant tumors. In various embodiments,the ovarian epithelial cancer is stage I (e.g., stage IA, IB, or IC),stage II (e.g., stage IIA, IIB, or ITC), stage III (e.g., stage IIIA,IIIB, or IIIC), or stage IV. In some embodiments, the individual may bea human who has a gene, genetic mutation, or polymorphism associatedwith ovarian cancer (e.g., BRCA1 or BRCA2) or has one or more extracopies of a gene associated with ovarian cancer (e.g., one or more extracopies of the HER2 gene). In some embodiments, the ovarian cancer is anovarian germ cell tumor. Exemplary histologic subtypes includedysgerminomas or other germ cell tumors (e.g., endodermal sinus tumorssuch as hepatoid or intestinal tumors, embryonal carcinomas,olyembryomas, choriocarcinomas, teratomas, or mixed form tumors).Exemplary teratomas are immature teratomas, mature teratomas, solidteratomas, and cystic teratomas (e.g., dermoid cysts such as maturecystic teratomas, and dermoid cysts with malignant transformation). Someteratomas are monodermal and highly specialized, such as struma ovarii,carcinoid, struma ovarii and carcinoid, or others (e.g., malignantneuroectodermal and ependymomas). In some embodiments, the ovarian germcell tumor is stage I (e.g., stage IA, IB, or IC), stage II (e.g., stageIIA, IIB, or ITC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stageIV.

The MASCT methods described herein in some embodiments are notapplicable to patients with cancers of T-cell origin, such as T-celllymphoma.

Several viruses are related to cancer in humans. For example, HepatitisB virus (HBV) can cause chronic infection of the liver, increasing anindividual's chance of liver cancer, or hepatocellular carcinoma (HCC).Human papilloma viruses (HPVs) are a group of more than 150 relatedviruses, which cause papilloma, or warts, when they infect and grow inskin or mucous membranes, such as the mouth, throat, or vagina. Severaltypes of HPV (including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, 59 and 6) are known to cause cervical cancer. HPVs also play a rolein inducing or causing other cancers of the genitalia, and are linked tosome cancers of the mouth and throat. Epstein-Barr virus (EBV) is a typeof herpes virus, which chronically infects and remains latent in Blymphocytes. EBV infection increases an individual's risk of developingnasopharyngeal carcinoma and certain types of fast-growing lymphomassuch as Burkitt lymphoma. EBV is also linked to Hodgkin lymphoma andsome cases of gastric cancer. In addition to causing cancer orincreasing risk of developing cancer, viral infections, such asinfections with HBV, HPV, and EBV, may result in damage to tissues ororgans, which can increase the disease burden of an individual sufferingfrom a cancer, and contribute to cancer progression.

It is known in the art that the human body can be induced to mounteffective and specific immune response, including cytotoxic T cellresponse, against several cancer-related viruses, such as HBV, HPV andEBV, including their various subtypes. Therefore, in some embodiments,there is provided a method of treating a virus-related cancer in anindividual, comprising administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing a population of T cells with a population of antigenpresenting cells (such as dendritic cells) loaded with a plurality oftumor antigen peptides. In some embodiments, the individual haspreviously been administered with an effective amount of antigenpresenting cells loaded with the plurality of tumor antigen peptides. Insome embodiments, the method further comprises administering to theindividual an effective amount of antigen presenting cells loaded with aplurality of tumor antigen peptides. In some embodiments, the virus isHBV, HPV, or EBV. In some embodiments, the cancer is HBV-relatedhepatocellular carcinoma, HPV-related cervical cancer, or EBV-relatednasopharyngeal carcinoma.

The methods described herein can be used for any one or more of thefollowing purposes: alleviating one or more symptoms of cancer, delayingprogression of cancer, shrinking cancer tumor size, disrupting (such asdestroying) cancer stroma, inhibiting cancer tumor growth, prolongingoverall survival, prolonging disease-free survival, prolonging time tocancer disease progression, preventing or delaying cancer tumormetastasis, reducing (such as eradiating) preexisting cancer tumormetastasis, reducing incidence or burden of preexisting cancer tumormetastasis, preventing recurrence of cancer, and/or improving clinicalbenefit of cancer.

APC, T Cell, and Tumor Antigen Peptide

The methods described herein in some embodiments use Antigen presentingcells (APCs) and activated T cells. APCs are cells of the immune systemthat are capable of activating T-cells. APCs include, but are notlimited to, certain macrophages, B cells, and dendritic cells (DCs).Dendritic Cells are members of a diverse population of morphologicallysimilar cell types found in lymphoid or non-lymphoid tissues. Thesecells are characterized by their distinctive morphology and highexpression levels of surface class I and class II MEW molecules, whichare proteins that present antigen peptides to the T cells. DCs, otherAPCs, and T cells can be isolated or derived (such as differentiated)from a number of tissue sources, and conveniently, from peripheralblood, such as the peripheral blood mononuclear cells (PBMCs) derivedfrom peripheral blood.

T cells, or T lymphocytes, play a central role in cell-mediatedimmunity. Each clone of activated T cells express a distinct T-cellreceptor (TCR) on the surface, which is responsible for recognizingantigens bound to MHC molecules on APCs and on target cells (such ascancer cells). T cells are subdivided into several types, eachexpressing a unique combination of surface proteins and each having adistinct function.

Cytotoxic T cells (TC) participate in the immune response to anddestruction of tumor cells and other infected cells, such as virusinfected cells. Generally, TC cells function by recognizing a class IMHC presented antigen on an APC or any target cell. Stimulation of theTCR, along with a co-stimulator (for example CD28 on the T cell bindingto B7 on the APC, or stimulation by a helper T cell), results inactivation of the TC cell. The activated TC cell can then proliferateand release cytotoxins, thereby destroying the APC, or a target cell(such as a cancer cell). Mature TC cells generally express surfaceproteins CD3 and CD8. Cytotoxic T cells belong to CD3⁺CD8⁺ T cells.

Helper T cells (TH) are T cells that help the activity of other immunecells by releasing T cell cytokines, which can regulate or suppressimmune responses, induce cytotoxic T cells, and maximize cell killingactivities of macrophages. Generally, TH cells function by recognizing aclass II MHC presented antigen on an APC. Mature TH cells express thesurface proteins CD3 and CD4. Helper T cells belong to CD3⁺CD4⁺ T cells.

Natural killer (NK) T cells are a heterogeneous group of T cells thatshare properties of both T cells and natural killer cells. Activation ofNK T cells results in production of pro-inflammatory cytokines,chemokines and cell factors. They express CD56, a surface moleculecommonly expressed on natural killer cells. NK T cells belong toCD3⁺CD56⁺ T cells.

Regulatory T cells (T_(REG) cells) generally modulate the immune systemby promoting tolerance for self-antigens, thereby limiting autoimmuneactivity. In cancer immunotherapy, T_(REG) contributes to escape of thecancer cells from the immune response. T_(REG) cells generally expressCD3, CD4, CD7, CD25, CTLA4, GITR, GARP, FOXP3, and/or LAP.CD4⁺CD25⁺Foxp3⁺ T cells are one class of T_(REG) cells.

Memory T cells (Tm) are T cells that have previously encountered andresponded to their specific antigens, or T cells that differentiatedfrom activated T cells. Although tumor specific Tms constitutes a smallproportion of the total T cell amount, they serve critical functions insurveillance of tumor cells during a person's entire lifespan. If tumorspecific Tms encounter tumor cells expressing their specific tumorantigens, the Tms are immediately activated and clonally expanded. Theactivated and expanded T cells differentiate into effector T cells tokill tumor cells with high efficiency. Memory T cells are important forestablishing and maintaining long-term tumor antigen specific responsesof T cells.

Typically, an antigen for T cells is a protein molecule or a linearfragment of a protein molecule that can be recognized by a T-cellreceptor (TCR) to elicit specific T cell response. The antigen can bederived from a foreign source such as a virally encoded protein, or anendogenous source such as a protein expressed intracellularly or on thecell surface. The minimal fragment of an antigen that is directlyinvolved in interaction with a particular TCR is known as an epitope.Multiple epitopes can exist in a single antigen, wherein each epitope isrecognized by a distinct TCR encoded by a particular clone of T cells.

In order to be recognized by a TCR, an antigen peptide or antigenfragment can be processed into an epitope by an APC (such as a dendriticcell), and then bound in an extended conformation inside a MajorHistocompatibility (MHC) molecule to form an MHC-peptide complex on thesurface of an APC (such as a dendritic cell). MHC molecules in human arealso known as human leukocyte antigens (HLA). The MHC provides anenlarged binding surface for strong association between TCR and epitope,while a combination of unique amino acid residues within the epitopeensures specificity of interaction between TCR and the epitope. Thehuman MHC molecules are classified into two types—MHC class I and MHCclass II—based on their structural features, especially the length ofepitopes bound inside the corresponding MHC complexes. MHC-I epitopesare epitopes bound to and represented by an MHC class I molecule. MHC-IIepitopes are epitopes bound to and represented by an MHC class IImolecule. MHC-I epitopes are typically about 8 to about 11 amino acidslong, whereas MHC-II epitopes are about 13 to about 17 amino acids long.Due to genetic polymorphism, various subtypes exist for both MHC class Iand MHC class II molecules among the human population. T cell responseto a specific antigen peptide presented by an MHC class I or MHC classII molecule on an APC or a target cell is known as MHC-restricted T cellresponse.

Tumor antigen peptides are derived from tumor antigen proteins (alsoreferred to herein as “tumor antigens”) that are overexpressed in cancercells, but have little to no expression levels (such as less than aboutany of 10, 100, 1000, or 5000 copies per cell) in normal cells. Sometumor antigen peptides are derived from tumor-specific antigens (TSA),differentiation antigens, or overexpressed antigens (also known astumor-associated antigens, or TAAs). Some tumor antigen peptides arederived from mutant protein antigens that are only present in cancercells, but absent in normal cells.

Antigen Loading of Dendritic Cells

The present invention provides a method of preparing a population ofdendritic cells loaded with a plurality of tumor antigen peptides usefulfor eliciting MHC-restricted T cell response in an individual,comprising contacting a population of dendritic cells with a pluralityof tumor antigen peptides. Dendritic cells prepared by the method can beused in any embodiment of the MASCT methods described herein, or toprepare activated T cells or co-culture of dendritic cells and T cellsas described in the next section.

In some embodiments of the methods of preparing the multiple-antigenloaded dendritic cells, the population of dendritic cells is contactedwith more than about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50 tumor antigenpeptides. In some embodiments, the population of dendritic cells iscontacted with a plurality of tumor antigen peptides comprising at leastabout any of 1, 5, 10, 15, 20, 25, 30, 35 or 40 of epitopes selectedfrom the group consisting of SEQ ID NOs: 1-40. In some embodiments, thepopulation of dendritic cells is contacted with about any of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more tumor antigen peptidesselected from the group consisting of the tumor antigen peptides in FIG.2C and FIG. 29A. In some embodiments, the population of dendritic cellsis contacted with about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, or more tumor antigen peptides derived from proteins selectedfrom the group consisting of hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1,MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA1, KRAS, PARP4, MLL3,and MTHFR.

In some embodiments, the dendritic cells are mature dendritic cells thatpresent one or more tumor antigen peptides of the plurality of tumorantigen peptides. The mature dendritic cells prepared by any of themethods described herein may present about any one of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or50 tumor antigen peptides. Compared to naïve dendritic cells, ordendritic cells that have not been loaded with a plurality of tumorantigen peptides, the multiple-antigen loaded dendritic cells may haveenhanced level of presentation for more than about any of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40,or 50 tumor antigen peptides. In some embodiments, the mature dendriticcells have enhanced level of presentation of at least about any of 1, 5,10, 15, 20, 25, 30, 35, or 40 of epitopes selected from the groupconsisting of SEQ ID NOs: 1-40. In some embodiments, the maturedendritic cells have enhanced level of presentation for more than ten ofthe tumor antigen peptides. In some embodiments, the mature dendriticcells have enhanced level of presentation of about any of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or more tumor antigen peptides as shownin FIG. 2C and FIG. 29A. In some embodiments, the mature dendritic cellshave enhanced level of presentation of about any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or more tumor antigen peptides derived fromproteins selected from the group consisting of hTERT, p53, Survivin,NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp,CDCA1, KRAS, PARP4, MLL3, and MTHFR.

An exemplary embodiment of the contacting of a population of dendriticcells with a plurality of tumor antigen peptides comprises pulsing theplurality of tumor antigen peptides into the population of dendriticcells, such as immature dendritic cells, or dendritic cells contained inor derived (such as differentiated) from the PBMCs. As known in the art,pulsing refers to a process of mixing cells, such as dendritic cells,with a solution containing antigen peptides, and optionally subsequentlyremoving the antigen peptides from the mixture. The population ofdendritic cells may be contacted with a plurality of tumor antigenpeptides for seconds, minutes, or hours, such as about any of 30seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6days, one week, 10 days, or more. The concentration of each tumorantigen peptide used in the contacting step may be about any of 0.1,0.5, 1, 2, 3, 5, or 10 μg/mL. In some embodiments, the concentration ofthe tumor antigen peptides is about 0.1-200 μg/mL, including for exampleabout any of 0.1-0.5, 0.5-1, 1-10, 10-50, 50-100, 100-150, or 150-200μg/mL.

In some embodiments, the population of dendritic cells is contacted withthe plurality of tumor antigen peptides in the presence of a compositionthat facilitates the uptake of the plurality of tumor antigen peptidesby the dendritic cells. In some embodiments, compounds, materials orcompositions may be included in a solution of the plurality of tumorantigen peptides to facilitate peptide uptake by the dendritic cells.Compounds, materials or compositions that facilitate the uptake of theplurality of tumor antigen peptides by the dendritic cells include, butare not limited to, lipid molecules and peptides with multiplepositively charged amino acids. In some embodiments, more than about anyof 50%, 60%, 70%, 80%, 90%, or 95% of the tumor antigen peptides areuptaken by the population of dendritic cells. In some embodiments, morethan about any of 50%, 60%, 70%, 80%, 90%, or 95% of the dendritic cellsin the population uptake at least one tumor antigen peptide.

In some embodiments, there is provided a method of preparing apopulation of dendritic cells loaded with a plurality of tumor antigenpeptides, comprising contacting a population of immature dendritic cellswith a plurality of tumor antigen peptides. In some embodiments, themethod further comprises inducing maturation of the population ofimmature dendritic cells with a plurality of Toll-like Receptor (TLR)agonists. In some embodiments, the method comprises contacting thepopulation of immature dendritic cells with a plurality of TLR agonistsand a plurality of tumor antigen peptides to obtain a population ofmature dendritic cells loaded with the plurality of tumor antigenpeptides. Exemplary TLR agonists include, but are not limited to,polyIC, MALP and R848. Cytokines and other appropriate molecules may befurther included in the culturing media in the maturation step. Thepopulation of immature dendritic cells may be induced by TLR agonists tomature for at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or20 days. In some embodiments, the population of immature dendritic cellsis induced to mature for about 8 days.

Dendritic cells (such as immature dendritic cells) may be obtained fromvarious sources, including autologous sources, i.e. from the individualreceiving the treatment. A convenient source of dendritic cells is thePBMCs from the peripheral blood. For example, monocytes, a type of whiteblood cells, are abundant in PBMCs, comprising about 10-30% of totalPBMCs. Monocytes can be induced to differentiate into dendritic cells,such as immature dendritic cells, using cytokines. In some embodiments,the immature dendritic cells are prepared by obtaining a population ofPBMCs, obtaining a population of monocytes from the population of PBMCs,and contacting the population of monocytes with a plurality of cytokinesto obtain a population of immature dendritic cells. Exemplary cytokinesthat may be used to induce differentiation of monocytes include, but arenot limited to, GM-CSF and IL-4, with conditions (such asconcentrations, temperature, CO₂ level etc.) known in the art. Theadherent fraction of PBMCs contains the majority of monocytes in PBMCs.In some embodiments, the monocytes from the adherent fraction of PBMCsare contacted with cytokines to obtain a population of immaturedendritic cells. PBMCs can be conveniently obtained by centrifugation ofa sample of peripheral blood, or using apheresis methods to collect froman individual. In some embodiments, the population of PBMCs is obtainedby density gradient centrifugation of a sample of human peripheralblood. In some embodiments, the sample is from the individual thatreceives the multiple-antigen loaded dendritic cells, activated T cells,or other immunotherapeutic compositions prepared using themultiple-antigen loaded dendritic cells.

In some embodiments, there is provided a method of preparing apopulation of dendritic cells loaded with a plurality of tumor antigenpeptides useful for eliciting MHC-restricted T cell response in anindividual, comprising the steps of obtaining a population of peripheralblood mononuclear cells (PBMCs) from an individual, obtaining apopulation of monocytes from the population of PBMCs, obtaining apopulation of dendritic cells from the population of monocytes, andcontacting the population of dendritic cells with a plurality of tumorantigen peptides to obtain a population of dendritic cells loaded withthe plurality of tumor antigen peptides. In some embodiments, there isprovided a method of preparing a population of dendritic cells loadedwith a plurality of tumor antigen peptides useful for elicitingMHC-restricted T cell response in an individual, comprising the steps ofobtaining a population of PBMCs from an individual (such as theindividual), obtaining a population of monocytes from the population ofPBMCs, contacting the population of monocytes with a plurality ofcytokines (such as GM-CSF and IL-4) to obtain a population of immaturedendritic cells, and contacting the population of immature dendriticcells with a plurality of TLR agonists and a plurality of tumor antigenpeptides to obtain the population of dendritic cells loaded with theplurality of tumor antigen peptides.

Further provided by the present invention is an isolated population ofdendritic cells prepared by any of the embodiments of the methodsdescribed herein. In some embodiments, the isolated population ofdendritic cells is capable of eliciting MHC-restricted T cell responsein vivo or ex vivo. In some embodiments, the MHC-restricted T cellresponse is mediated by both MHC class I and MHC class II molecules. Insome embodiments, the isolated population of dendritic cells is capableof inducing differentiation and proliferation of tumor antigen-specificT cells.

Preparation of Activated T Cells

Further provided in the present invention is a method of preparing apopulation of activated T cells useful for treating a cancer in anindividual, comprising co-culturing a population of T cells with apopulation of antigen presenting cells (such as dendritic cells) loadedwith a plurality of tumor antigen peptides. Any embodiment of themultiple-antigen loaded dendritic cells in the previous section may beused to prepare the activated T cells. In some embodiments, thepopulation of T cells and the population of dendritic cells are derivedfrom the same individual, such as an individual with a cancer (e.g. lowto moderate grade cancer). In some embodiments, the population of Tcells, the population of dendritic cells, or both is derived fromautologous sources, i.e. from the individual that receives the activatedT cells, the multiple-antigen loaded dendritic cells, or both.

In some embodiments, the population of T cells and the population ofdendritic cells loaded with the plurality of tumor antigen peptides areco-cultured for at least about any of 2, 4, 6, 8, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, or 30 days. In some embodiments, the population of Tcells is co-cultured with the population of dendritic cells loaded withthe plurality of tumor antigen peptides for about 7 days to about 21days (such as about 7 days to about 14 days, about 7 days to about 10days, about 10 days to about 15 days, about 14 days to about 21 days,about 10 days, 14 days, 16 days, 18 days, or 21 days). In someembodiments, the population of T cells is co-cultured with thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides for about 10 days. In some embodiments, the population of Tcells is co-cultured with the population of dendritic cells loaded withthe plurality of tumor antigen peptides for about 14 days.

The population of T cells used in any embodiment of the methodsdescribed herein may be derived from a variety of sources. A convenientsource of T cells is from the PBMCs of the human peripheral blood. Thepopulation of T cells may be isolated from the PBMCs, or alternatively,a population of PBMCs enriched with T cells (such as by addition of Tcell specific antibodies and cytokines) can be used in the co-culture.In some embodiments, the population of T cells used in the co-culture isobtained from the non-adherent fraction of peripheral blood mononuclearcells (PBMCs). In some embodiments, the PBMCs are obtained by densitygradient centrifugation of a sample of peripheral blood. In someembodiments, the population of T cells is obtained by culturing thenon-adherent fraction of PBMCs with at least one cytokine (such as IL-2)with or without an anti-CD3 antibody (such as OKT3) (a process referredherein as “maintaining T cells”). In some embodiments, the non-adherentfraction of PBMCs is cultured in the presence of an immune checkpointinhibitor. In some embodiments, the immune checkpoint inhibitor is aninhibitor of an immune checkpoint molecule selected from the groupconsisting of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3.The non-adherent fraction of PBMCs may be cultured for at least aboutany of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. Insome embodiments, the population of activated T cells is prepared byobtaining a population of non-adherent PBMCs, and co-culturing thepopulation of non-adherent PBMCs with a population of dendritic cellsloaded with a plurality of tumor antigen peptides (such as in thepresence of at least one cytokine (such as IL-2) and optionally ananti-CD3 antibody, and optionally an immune checkpoint inhibitor).

The co-culture may further include cytokines and other compounds tofacilitate activation, maturation, and/or proliferation of the T cells,as well as to prime T cells for later differentiation into memory Tcells. Exemplary cytokines that may be used in this step include, butare not limited to, IL-7, IL-15, IL-21 and the like. Certain cytokinesmay help suppress the percentage of T_(REG) in the population ofactivated T cells in the co-culture. For example, in some embodiments, ahigh dose (such as at least about any of 200, 300, 400, 500, 600, 700,800, 900, 1000, 1200, or 1500 U/ml) of a cytokine (such as IL-2) is usedto co-culture the population of T cells and the population of dendriticcells loaded with the plurality of tumor antigen peptides to obtain apopulation of activated T cells with a low percentage of T_(REG) cells.

The co-culture may also include one or more (such as any of 1, 2, 3, ormore) immune checkpoint inhibitors. In some embodiments, the populationof T cells is contacted with an immune checkpoint inhibitor prior to theco-culturing. For example, the population of T cells may be isolated Tcells, or T cells present in a mixture of cells, such as non-adherentfraction of PBMCs. In some embodiments, the population of non-adherentPBMCs are contacted with an immune checkpoint inhibitor prior to theco-culturing. In some embodiments, the population of T cells ornon-adherent PBMCs are contacted with the immune checkpoint inhibitorfor at least about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, or moredays. In some embodiments, the population of T cells or non-adherentPBMCs are contacted with the immune checkpoint inhibitor for about 5days to about 14 days. In some embodiments, the PBMCs are contacted withthe immune checkpoint inhibitor for about 8 days.

In some embodiments, the population of T cells is co-cultured with thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides in the presence of an immune checkpoint inhibitor. In someembodiments, the immune checkpoint inhibitor is an inhibitor of aninhibitory checkpoint molecule selected from the group consisting ofPD-1, PD-L1, PD-L2, CTLA-4, BLTA, TIM-3, and LAG-3. In some embodiments,the immune checkpoint inhibitor is an inhibitor of PD-1. In someembodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody,such as nivolumab (for example, OPDIVO®), Pembrolizumab (for example,KEYTRUDA®) or SHR-1210. In some embodiments, the immune checkpointinhibitor is an inhibitor of PD-L1. In some embodiments, the immunecheckpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, theimmune checkpoint inhibitor is an inhibitor of CTLA-4. In someembodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody,such as Ipilimumab (for example, YERVOY®).

The population of T cells may be stimulated with the population of DCsloaded with the plurality of tumor antigen peptides for any number oftimes, such as any of 1, 2, 3, or more times. In some embodiments, thepopulation of T cells is stimulated once. In some embodiments, thepopulation of T cells is stimulated for at least two times. In someembodiments, for each stimulation, a population of DCs loaded with theplurality of tumor antigen peptides is added to the co-culture. Thepopulation of DCs may be freshly prepared and pulsed with the pluralityof tumor antigen peptides, or may be obtained from a stock of thepopulation of DCs prepared for the initial stimulation.

Accordingly, there is provided a method of preparing a population ofactivated T cells, comprising: (a) preparing a population of dendriticcells loaded with a plurality of tumor antigen peptides; and (b)co-culturing a population of dendritic cells loaded with the pluralityof tumor antigen peptides and a population of non-adherent PBMCs toobtain the population of activated T cells, wherein the population ofdendritic cells and the population of non-adherent PBMCs are obtainedfrom a population of PBMCs from an individual. In some embodiments, theco-culturing is in the presence of a plurality of cytokines (such asIL-2, IL-7, IL-15, IL-21 or any combination thereof). In someembodiments, the co-culturing is in the presence of an anti-CD3 antibody(such as OKT3) and a plurality of cytokines (such as IL-2, IL-7, IL-15,IL-21 or any combination thereof). In some embodiments, the populationof non-adherent PBMCs is contacted with an immune checkpoint inhibitor(such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA,or LAG-3) prior to and/or during the co-culturing. In some embodiments,the method further comprises obtaining the population of PBMCs from theindividual.

In some embodiments, there is provided a method of preparing apopulation of activated T cells, comprising: (a) inducingdifferentiation of a population of monocytes into a population ofdendritic cells (such as in the presence of GM-CSF and IL-4); (b)contacting the population of dendritic cells with a plurality of tumorantigen peptides to obtain a population of dendritic cells loaded withthe plurality of tumor antigen peptides; and (c) co-culturing thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells, wherein the population of monocytes and thepopulation of non-adherent PBMCs are obtained from a population of PBMCsfrom an individual. In some embodiments, the population of dendriticcells loaded with the plurality of tumor antigen peptides is contactedwith a plurality of TLR agonists to induce maturation of the populationof dendritic cells loaded with the plurality of tumor antigen peptides.In some embodiments, the co-culturing is in the presence of a pluralityof cytokines (such as IL-2, IL-7, IL-15, IL-21 or any combinationthereof). In some embodiments, the co-culturing is in the presence of ananti-CD3 antibody (such as OKT3) and a plurality of cytokines (such asIL-2, IL-7, IL-15, IL-21 or any combination thereof). In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) prior to and/or during theco-culturing. In some embodiments, the method further comprises any oneor combination of the steps: (i) obtaining the population of PBMCs fromthe individual; (ii) obtaining the population of monocytes from thepopulation of PBMCs; and (iii) obtaining the population of non-adherentPBMCs from the population of PBMCs.

In some embodiments, there is provided a method of preparing apopulation of activated T cells, the method comprising obtaining apopulation of peripheral blood mononuclear cells (PBMCs) from anindividual, obtaining a population of monocytes from the population ofPBMCs, inducing differentiation of the population of monocytes into apopulation of dendritic cells (such as in the presence of GM-CSF andIL-4), contacting the population of immature dendritic cells with aplurality of Toll-like Receptor (TLR) agonists and a plurality of tumorantigen peptides to obtain a population of mature dendritic cells loadedwith the plurality of tumor antigen peptides, obtaining a population ofnon-adherent PBMCs from the population of PBMCs, and co-culturing thepopulation of mature dendritic cells loaded with the plurality of tumorantigen peptides and the population of non-adherent PBMCs in thepresence of a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21or any combination thereof), optionally an anti-CD3 antibody (such asOKT3), and optionally an immune checkpoint inhibitor (such as aninhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) toobtain the population of activated T cells.

Further provided by the present invention is an isolated population ofactivated T cells prepared by any embodiment of the methods describedherein. Also provided herein is a co-culture useful for treating cancerin an individual, comprising a population of T cells and a population ofdendritic cells loaded with a plurality of tumor antigen peptides. Insome embodiments of the co-culture, the population of T cells and thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides are derived from the same individual, such as the individualbeing treated. In some embodiments of the co-culture, the population ofmultiple-antigen loaded dendritic cells is prepared by any embodiment ofthe methods of preparation as described in the previous section, such aspulsing a plurality of tumor antigen peptides into a population ofdendritic cells, or contacting a population of dendritic cells with aplurality of tumor antigen peptides in the presence of a composition(such as lipid molecules, or peptides with multiple positively chargedamino acids) that facilitates the uptake of the plurality of tumorantigen peptides by the dendritic cells. The isolated population ofactivated T cells and the co-culture described in this section may beused in any embodiment of the MASCT methods. Immunotherapeuticcompositions comprising the isolated population of activated T cells orthe co-culture are useful for treating cancer, preventing tumorprogression or metastasis, or reducing cancer immune escape are providedherein. The isolated population of activated T cells and the co-culturemay also be used in the manufacture of a medicament for treating cancer,preventing tumor progression or metastasis, or reducing cancer immuneescape.

It is intended that any of the steps and parameters described herein forpreparing a population of dendritic cells loaded with a plurality oftumor antigen peptides or for preparing a population of activated Tcells can be combined with any of the steps and parameters describedherein for the MASCT method, as if each and every combination isindividually described.

For example, in some embodiments, there is provided an isolatedpopulation of activated T cells prepared by co-culturing a population ofT cells with a population of dendritic cells loaded with a plurality oftumor antigen peptides. In some embodiments, the population of dendriticcells is prepared by contacting a population of dendritic cells with aplurality of tumor antigen peptides (such as in the presence of acomposition that facilitates the uptake of the plurality of tumorantigen peptides by the dendritic cells). In some embodiments, thepopulation of T cells is contacted with an immune checkpoint inhibitor(such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA,or LAG-3) prior to and/or during the co-culturing. In some embodiments,the population of dendritic cells, and the population of T cells arefrom the same source (such as the individual receiving the activated Tcells for treatment).

In some embodiments, there is provided an isolated population ofactivated T cells prepared by: (a) inducing differentiation of apopulation of monocytes into a population of dendritic cells (such as inthe presence of GM-CSF and IL-4); (b) contacting the population ofdendritic cells with a plurality of tumor antigen peptides to obtain apopulation of dendritic cells loaded with the plurality of tumor antigenpeptides; and (c) co-culturing the population of dendritic cells loadedwith the plurality of tumor antigen peptides and a population ofnon-adherent PBMCs, wherein the population of monocytes and thepopulation of non-adherent PBMCs are obtained from a population of PBMCsfrom an individual. In some embodiments, the population of dendriticcells loaded with the plurality of tumor antigen peptides is contactedwith a plurality of TLR agonists to induce maturation of the populationof dendritic cells loaded with the plurality of tumor antigen peptides.In some embodiments, the co-culturing is in the presence of a pluralityof cytokines (such as IL-2, IL-7, IL-15, IL-21 or any combinationthereof) and optionally an anti-CD3 antibody (such as OKT3). In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) prior to and/or during theco-culturing. In some embodiments, the method further comprises any oneor combination of the steps: (i) obtaining the population of PBMCs fromthe individual; (ii) obtaining the population of monocytes from thepopulation of PBMCs; and (iii) obtaining the population of non-adherentPBMCs from the population of PBMCs.

PBMC-Based MASCT

A variation of the MASCT method, named PBMC-based MASCT, directly usesPBMCs, which comprise APCs and T cells, without isolating or derivingthe APCs (such as dendritic cells) or T cells for use in treating acancer in an individual.

Accordingly, in some embodiments, there is provided a method of treatinga cancer in an individual, comprising contacting a population ofperipheral blood mononuclear cells (PBMCs) with a plurality of tumorantigen peptides to obtain a population of activated PBMCs, andadministering to the individual an effective amount of the activatedPBMCs. In some embodiments, the population of PBMCs is contacted withthe plurality of tumor antigen peptides in the presence of a compositionthat facilitates the uptake of the plurality of tumor antigen peptidesby antigen presenting cells (such as dendritic cells) in the PBMCs. Insome embodiments, the population of PBMCs is contacted with theplurality of tumor antigen peptides in the presence of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, and LAG-3. In some embodiments, the population ofactivated PBMCs is contacted with IL-2. In some embodiments, theactivated PBMCs are administered for at least three times. In someembodiments, the interval between each administration of the activatedPBMCs is about 2 weeks to about 5 months (such as about 3 months). Insome embodiments, the activated PBMCs are administered intravenously. Insome embodiments, the population of PBMCs is obtained from theindividual being treated.

The PBMC-based MASCT method is suitable to treat any cancer (includingdifferent type or stages) that can be treated by the other embodimentsof the MASCT method as described in the previous sections. In someembodiments of the PBMC-based MASCT method, the cancer is selected fromthe group consisting of hepatic cellular carcinoma, cervical cancer,lung cancer, colorectal cancer, lymphoma, renal carcinoma, breastcancer, pancreatic cancer, gastric cancer, esophageal cancer, ovariancancer, prostate cancer, nasopharyngeal carcinoma, melanoma and braincancer.

In some embodiments, the PBMCs are autologous, i.e. obtained from theindividual being treated. In some embodiments, the peripheral blood fromthe individual has a low number of dendritic cells or T cells. In someembodiments, the PBMCs are contacted with cytokines, such as IL-2,GM-CSF, or the like, to induce differentiation, maturation, orproliferation of certain cells (such as dendritic cells, T cells, orcombination thereof) in the PBMCs concurrently or after the contactingstep. In some embodiments, the plurality of tumor antigen peptides isremoved after the contacting step. In some embodiments, the PBMCs arecontacted with the plurality of tumor antigen peptides for at leastabout any of 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 5hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, one week, 10 days,or more. In some embodiments, the PBMCs are contacted with the cytokinesfor at least about any of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, or 30 days. In some embodiments, the PBMCs are contacted withthe cytokines for about 14-21 days. In some embodiments, the PBMCs arecontacted with the cytokines for about 14 days.

In any of the PBMC-based MASCT methods above, the PBMCs are contactedwith one or more immune checkpoint inhibitors. In some embodiments, thepopulation of PBMCs is contacted with the plurality of tumor antigenpeptides in the presence of an immune checkpoint inhibitor. In someembodiments, the PBMCs are contacted with the immune checkpointinhibitor for at least about any of 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, or 30 days. In some embodiments, the PBMCs are contactedwith the immune checkpoint inhibitor for about 14 days to about 21 days.

Combination Therapy with Immune Checkpoint Inhibitor

The methods described herein for treating cancer can be used inmonotherapy as well as in combination therapy with another agent. Forexample, any of the MASCT methods (including the PBMC-based MASCTmethods) described herein may be combined with administration of one ormore (such as any of 1, 2, 3, 4, or more) immune checkpoint inhibitors.

Thus, in some embodiments, there is provided a method of treating acancer in an individual comprising: (a) optionally administering to theindividual an effective amount of dendritic cells loaded with aplurality of tumor antigen peptides; (b) administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof dendritic cells loaded with the plurality of tumor antigen peptides;and (c) administering to the individual an effective amount of an immunecheckpoint inhibitor. In some embodiments, the activated T cells and theimmune checkpoint inhibitor are administered simultaneously, such as inthe same composition. In some embodiments, the activated T cells and theimmune checkpoint inhibitor are administered sequentially. In someembodiments, the interval between the administration of the dendriticcells and the administration of the activated T cells is about 7 days toabout 21 days (such as about 7 days to about 14 days, about 14 days toabout 21 days, about 10 days or about 14 days). In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, thepopulation of T cells is co-cultured with the population of dendriticcells loaded with the plurality of tumor antigen peptides for about 7days to about 21 days (such as about 7 days to about 14 days, about 14days to about 21 days, or about 10 days). In some embodiments, thepopulation of T cells is derived from the non-adherent portion of apopulation of peripheral blood mononuclear cells (PBMCs). In someembodiments, the co-culturing further comprises contacting the activatedT cells with a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21,or any combination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of T cells is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3) prior to and/or during the co-culturing.In some embodiments, the population of dendritic cells loaded with theplurality of tumor antigen peptides is prepared by contacting apopulation of dendritic cells with the plurality of tumor antigenpeptides. In some embodiments, the population of T cells and thepopulation of dendritic cells are derived from the same individual. Insome embodiments, the population of T cells, the population of dendriticcells, the population of PBMCs, or any combination thereof is derivedfrom the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells; (e) administering to the individual an effective amount of theactivated T cells; and (f) administering to the individual an effectiveamount of an immune checkpoint inhibitor, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs. In some embodiments, the activated T cells and theimmune checkpoint inhibitor are administered simultaneously, such as inthe same composition. In some embodiments, the activated T cells and theimmune checkpoint inhibitor are administered sequentially. In someembodiments, the interval between the administration of the dendriticcells and the administration of the activated T cells is about 7 days toabout 21 days (such as about 7 days to about 14 days, about 14 days toabout 21 days, about 10 days or about 14 days). In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, or about 10 days). Insome embodiments, the co-culturing further comprises contacting theactivated T cells with a plurality of cytokines (such as IL-2, IL-7,IL-15, IL-21, or any combination thereof) and optionally an anti-CD3antibody. In some embodiments, the population of non-adherent PBMCs iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of PBMCs is obtained from the individualbeing treated.

In some embodiments, there is provided a method of treating a cancer inan individual comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs; (b) administering to the individual an effective amount of theactivated PBMCs; and (c) administering to the individual an effectiveamount of an immune checkpoint inhibitor. In some embodiments, theactivated PBMCs and the immune checkpoint inhibitor are administeredsimultaneously, such as in the same composition. In some embodiments,the activated PBMCs and the immune checkpoint inhibitor are administeredsequentially. In some embodiments, the PBMCs is contacted with theplurality of tumor antigen peptides in the presence of a compositionthat facilitates the uptake of the plurality of tumor antigen peptidesby antigen presenting cells (such as dendritic cells) in the PBMCs. Insome embodiments, the population of activated PBMCs is further contactedwith IL-2. In some embodiments, the population of PBMCs is contactedwith the plurality of tumor antigen peptides in the presence of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3. In some embodiments, theactivated PBMCs are administered for at least three times. In someembodiments, the interval between each administration of the activatedPBMCs is about 2 weeks to about 5 months (such as about 3 months). Insome embodiments, the activated PBMCs are administered intravenously. Insome embodiments, the population of PBMCs is obtained from theindividual being treated.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-1. In some embodiments, the immune checkpoint inhibitor is ananti-PD-1 antibody. Exemplary anti-PD-1 antibodies include, but are notlimited to, Nivolumab, pembrolizumab, pidilizumab, BMS-936559, andatezolizumab, Pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, andTSR-042. In some embodiments, the immune checkpoint inhibitor isnivolumab (for example, OPDIVO®). In some embodiments, the immunecheckpoint inhibitor is Pembrolizumab (for example, KEYTRUDA®). In someembodiments, the immune checkpoint inhibitor is SHR-1210.

Thus, in some embodiments, there is provided a method of treating acancer in an individual comprising: (a) optionally administering to theindividual an effective amount of dendritic cells loaded with aplurality of tumor antigen peptides; (b) administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof dendritic cells loaded with the plurality of tumor antigen peptides;and (c) administering to the individual an effective amount of aninhibitor of PD-1. In some embodiments, the inhibitor of PD-1 is ananti-PD-1 antibody. In some embodiments, the inhibitor of PD-1 isselected from the group consisting of nivolumab, pembrolizumab, andSHR-1210. In some embodiments, the activated T cells and the inhibitorof PD-1 are administered simultaneously, such as in the samecomposition. In some embodiments, the activated T cells and theinhibitor of PD-1 are administered sequentially. In some embodiments,the interval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, thepopulation of T cells is co-cultured with the population of dendriticcells loaded with the plurality of tumor antigen peptides for about 7days to about 21 days (such as about 7 days to about 14 days, about 14days to about 21 days, or about 10 days). In some embodiments, thepopulation of T cells is derived from the non-adherent portion of apopulation of peripheral blood mononuclear cells (PBMCs). In someembodiments, the co-culturing further comprises contacting the activatedT cells with a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21,or any combination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of T cells is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1) prior to and/orduring the co-culturing. In some embodiments, the population ofdendritic cells loaded with the plurality of tumor antigen peptides isprepared by contacting a population of dendritic cells with theplurality of tumor antigen peptides. In some embodiments, the populationof T cells and the population of dendritic cells are derived from thesame individual. In some embodiments, the population of T cells, thepopulation of dendritic cells, the population of PBMCs, or anycombination thereof is derived from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells; (e) administering to the individual an effective amount of theactivated T cells; and (f) administering to the individual an effectiveamount of an inhibitor of PD-1, wherein the population of monocytes andthe population of non-adherent PBMCs are obtained from a population ofPBMCs. In some embodiments, the inhibitor of PD-1 is an anti-PD-1antibody. In some embodiments, the inhibitor of PD-1 is selected fromthe group consisting of nivolumab, pembrolizumab, and SHR-1210. In someembodiments, the activated T cells and the inhibitor of PD-1 areadministered simultaneously, such as in the same composition. In someembodiments, the activated T cells and the inhibitor of PD-1 areadministered sequentially. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the co-culturing is for about 7 daysto about 21 days (such as about 7 days to about 14 days, about 14 daysto about 21 days, or about 10 days). In some embodiments, theco-culturing further comprises contacting the activated T cells with aplurality of cytokines (such as IL-2, IL-7, IL-15, IL-21, or anycombination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1) prior toand/or during the co-culturing. In some embodiments, the population ofPBMCs is obtained from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs; (b) administering to the individual an effective amount of theactivated PBMCs; and (c) administering to the individual an effectiveamount of an inhibitor of PD-1. In some embodiments, the inhibitor ofPD-1 is an anti-PD-1 antibody. In some embodiments, the inhibitor ofPD-1 is selected from the group consisting of nivolumab, pembrolizumab,and SHR-1210. In some embodiments, the activated PBMCs and the inhibitorof PD-1 are administered simultaneously, such as in the samecomposition. In some embodiments, the activated PBMCs and the inhibitorof PD-1 are administered sequentially. In some embodiments, the PBMCs iscontacted with the plurality of tumor antigen peptides in the presenceof a composition that facilitates the uptake of the plurality of tumorantigen peptides by antigen presenting cells (such as dendritic cells)in the PBMCs. In some embodiments, the population of activated PBMCs isfurther contacted with IL-2. In some embodiments, the population ofPBMCs is contacted with the plurality of tumor antigen peptides in thepresence of an immune checkpoint inhibitor, such as an inhibitor ofPD-1. In some embodiments, the activated PBMCs are administered for atleast three times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual comprising: (a) optionally administering to the individualan effective amount of dendritic cells loaded with a plurality of tumorantigen peptides; (b) administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing a population of T cells with a population of dendriticcells loaded with the plurality of tumor antigen peptides; and (c)administering to the individual an effective amount of pembrolizumab(such as KEYTRUDA®). In some embodiments, the activated T cells and thepembrolizumab are administered simultaneously, such as in the samecomposition. In some embodiments, the activated T cells and thepembrolizumab are administered sequentially. In some embodiments, thepembrolizumab is administered intravenously (such as by infusion forover about 30 minutes). In some embodiments, the pembrolizumab isadministered at about 2 mg/kg. In some embodiments, the pembrolizumab isadministered about once every 3 weeks. In some embodiments, the intervalbetween the administration of the dendritic cells and the administrationof the activated T cells is about 7 days to about 21 days (such as about7 days to about 14 days, about 14 days to about 21 days, about 10 daysor about 14 days). In some embodiments, the dendritic cells loaded withthe plurality of tumor antigen peptides are administered subcutaneously.In some embodiments, the dendritic cells loaded with the plurality oftumor antigen peptides are administered for at least three times. Insome embodiments, the activated T cells are administered intravenously.In some embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,or about 10 days). In some embodiments, the population of T cells isderived from the non-adherent portion of a population of peripheralblood mononuclear cells (PBMCs). In some embodiments, the co-culturingfurther comprises contacting the activated T cells with a plurality ofcytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof)and optionally an anti-CD3 antibody. In some embodiments, the populationof T cells is contacted with an immune checkpoint inhibitor (such as aninhibitor of PD-1) prior to and/or during the co-culturing. In someembodiments, the population of dendritic cells loaded with the pluralityof tumor antigen peptides is prepared by contacting a population ofdendritic cells with the plurality of tumor antigen peptides. In someembodiments, the population of T cells and the population of dendriticcells are derived from the same individual. In some embodiments, thepopulation of T cells, the population of dendritic cells, the populationof PBMCs, or any combination thereof is derived from the individualbeing treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells; (e) administering to the individual an effective amount of theactivated T cells; and (f) administering to the individual an effectiveamount of pembrolizumab (such as KEYTRUDA®), wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs. In some embodiments, the activated T cells and thepembrolizumab are administered simultaneously, such as in the samecomposition. In some embodiments, the activated T cells and thepembrolizumab are administered sequentially. In some embodiments, thepembrolizumab is administered intravenously (such as by infusion forover about 30 minutes). In some embodiments, the pembrolizumab isadministered at about 2 mg/kg. In some embodiments, the pembrolizumab isadministered about once every 3 weeks. In some embodiments, the intervalbetween the administration of the dendritic cells and the administrationof the activated T cells is about 7 days to about 21 days (such as about7 days to about 14 days, about 14 days to about 21 days, about 10 daysor about 14 days). In some embodiments, the dendritic cells loaded withthe plurality of tumor antigen peptides are administered subcutaneously.In some embodiments, the dendritic cells loaded with the plurality oftumor antigen peptides are administered for at least three times. Insome embodiments, the activated T cells are administered intravenously.In some embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the co-culturing is for about 7 daysto about 21 days (such as about 7 days to about 14 days, about 14 daysto about 21 days, or about 10 days). In some embodiments, theco-culturing further comprises contacting the activated T cells with aplurality of cytokines (such as IL-2, IL-7, IL-15, IL-21, or anycombination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1) prior toand/or during the co-culturing. In some embodiments, the population ofPBMCs is obtained from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs; (b) administering to the individual an effective amount of theactivated PBMCs; and (c) administering to the individual an effectiveamount of pembrolizumab (such as KEYTRUDA®). In some embodiments, theactivated PBMCs and the pembrolizumab are administered simultaneously,such as in the same composition. In some embodiments, the activatedPBMCs and the pembrolizumab are administered sequentially. In someembodiments, the pembrolizumab is administered intravenously (such as byinfusion for over about 30 minutes). In some embodiments, thepembrolizumab is administered at about 2 mg/kg. In some embodiments, thepembrolizumab is administered about once every 3 weeks. In someembodiments, the PBMCs is contacted with the plurality of tumor antigenpeptides in the presence of a composition that facilitates the uptake ofthe plurality of tumor antigen peptides by antigen presenting cells(such as dendritic cells) in the PBMCs. In some embodiments, thepopulation of activated PBMCs is further contacted with IL-2. In someembodiments, the population of PBMCs is contacted with the plurality oftumor antigen peptides in the presence of an immune checkpointinhibitor, such as an inhibitor of PD-1. In some embodiments, theactivated PBMCs are administered for at least three times. In someembodiments, the interval between each administration of the activatedPBMCs is about 2 weeks to about 5 months (such as about 3 months). Insome embodiments, the activated PBMCs are administered intravenously. Insome embodiments, the population of PBMCs is obtained from theindividual being treated.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is ananti-PD-L1 antibody. Exemplary anti-PD-L1 antibodies include, but arenot limited to, KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A,MEDI4736, Avelumab, or STI-A1010.

Thus, in some embodiments, there is provided a method of treating acancer in an individual comprising: (a) optionally administering to theindividual an effective amount of dendritic cells loaded with aplurality of tumor antigen peptides; (b) administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof dendritic cells loaded with the plurality of tumor antigen peptides;and (c) administering to the individual an effective amount of aninhibitor of PD-L1. In some embodiments, the inhibitor of PD-L1 is ananti-PD-L1 antibody. In some embodiments, the activated T cells and theinhibitor of PD-L1 are administered simultaneously, such as in the samecomposition. In some embodiments, the activated T cells and theinhibitor of PD-L1 are administered sequentially. In some embodiments,the interval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, thepopulation of T cells is co-cultured with the population of dendriticcells loaded with the plurality of tumor antigen peptides for about 7days to about 21 days (such as about 7 days to about 14 days, about 14days to about 21 days, or about 10 days). In some embodiments, thepopulation of T cells is derived from the non-adherent portion of apopulation of peripheral blood mononuclear cells (PBMCs). In someembodiments, the co-culturing further comprises contacting the activatedT cells with a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21,or any combination thereof) and optionally an anti-CD3 antibody. In someembodiments, the population of T cells is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-L1) prior to and/orduring the co-culturing. In some embodiments, the population ofdendritic cells loaded with the plurality of tumor antigen peptides isprepared by contacting a population of dendritic cells with theplurality of tumor antigen peptides. In some embodiments, the populationof T cells and the population of dendritic cells are derived from thesame individual. In some embodiments, the population of T cells, thepopulation of dendritic cells, the population of PBMCs, or anycombination thereof is derived from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells; (e) administering to the individual an effective amount of theactivated T cells; and (f) administering to the individual an effectiveamount of an inhibitor of PD-L1, wherein the population of monocytes andthe population of non-adherent PBMCs are obtained from a population ofPBMCs. In some embodiments, the inhibitor of PD-L1 is an anti-PD-L1antibody. In some embodiments, the activated T cells and the inhibitorof PD-L1 are administered simultaneously, such as in the samecomposition. In some embodiments, the activated T cells and theinhibitor of PD-L1 are administered sequentially. In some embodiments,the interval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, or about 10 days). Insome embodiments, the co-culturing further comprises contacting theactivated T cells with a plurality of cytokines (such as IL-2, IL-7,IL-15, IL-21, or any combination thereof) and optionally an anti-CD3antibody. In some embodiments, the population of non-adherent PBMCs iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-L1) prior to and/or during the co-culturing. In some embodiments, thepopulation of PBMCs is obtained from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs; (b) administering to the individual an effective amount of theactivated PBMCs; and (c) administering to the individual an effectiveamount of an inhibitor of PD-L1. In some embodiments, the inhibitor ofPD-L1 is an anti-PD-L1 antibody. In some embodiments, the activatedPBMCs and the inhibitor of PD-L1 are administered simultaneously, suchas in the same composition. In some embodiments, the activated PBMCs andthe inhibitor of PD-L1 are administered sequentially. In someembodiments, the PBMCs is contacted with the plurality of tumor antigenpeptides in the presence of a composition that facilitates the uptake ofthe plurality of tumor antigen peptides by antigen presenting cells(such as dendritic cells) in the PBMCs. In some embodiments, thepopulation of activated PBMCs is further contacted with IL-2. In someembodiments, the population of PBMCs is contacted with the plurality oftumor antigen peptides in the presence of an immune checkpointinhibitor, such as an inhibitor of PD-L1. In some embodiments, theactivated PBMCs are administered for at least three times. In someembodiments, the interval between each administration of the activatedPBMCs is about 2 weeks to about 5 months (such as about 3 months). Insome embodiments, the activated PBMCs are administered intravenously. Insome embodiments, the population of PBMCs is obtained from theindividual being treated.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCTLA-4. In some embodiments, the immune checkpoint inhibitor is ananti-CTLA-4 antibody. Exemplary anti-CTLA-4 antibodies include, but arenot limited to, Ipilimumab, Tremelimumab, and KAHR-102. In someembodiments, the immune checkpoint inhibitor is Ipilimumab (for example,YERVOY®).

Thus, in some embodiments, there is provided a method of treating acancer in an individual comprising: (a) optionally administering to theindividual an effective amount of dendritic cells loaded with aplurality of tumor antigen peptides; (b) administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof dendritic cells loaded with the plurality of tumor antigen peptides;and (c) administering to the individual an effective amount of aninhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is ananti-CTLA-4 antibody, such as Ipilimumab. In some embodiments, theactivated T cells and the inhibitor of CTLA-4 are administeredsimultaneously, such as in the same composition. In some embodiments,the activated T cells and the inhibitor of CTLA-4 are administeredsequentially. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of T cells isco-cultured with the population of dendritic cells loaded with theplurality of tumor antigen peptides for about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,or about 10 days). In some embodiments, the population of T cells isderived from the non-adherent portion of a population of peripheralblood mononuclear cells (PBMCs). In some embodiments, the co-culturingfurther comprises contacting the activated T cells with a plurality ofcytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof)and optionally an anti-CD3 antibody. In some embodiments, the populationof T cells is contacted with an immune checkpoint inhibitor (such as aninhibitor of CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of dendritic cells loaded with the pluralityof tumor antigen peptides is prepared by contacting a population ofdendritic cells with the plurality of tumor antigen peptides. In someembodiments, the population of T cells and the population of dendriticcells are derived from the same individual. In some embodiments, thepopulation of T cells, the population of dendritic cells, the populationof PBMCs, or any combination thereof is derived from the individualbeing treated.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells; (e) administering to the individual an effective amount of theactivated T cells; and (f) administering to the individual an effectiveamount of an inhibitor of CTLA-4, wherein the population of monocytesand the population of non-adherent PBMCs are obtained from a populationof PBMCs. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4antibody, such as Ipilimumab. In some embodiments, the activated T cellsand the inhibitor of CTLA-4 are administered simultaneously, such as inthe same composition. In some embodiments, the activated T cells and theinhibitor of CTLA-4 are administered sequentially. In some embodiments,the interval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, or about 10 days). Insome embodiments, the co-culturing further comprises contacting theactivated T cells with a plurality of cytokines (such as IL-2, IL-7,IL-15, IL-21, or any combination thereof) and optionally an anti-CD3antibody. In some embodiments, the population of non-adherent PBMCs iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofCTLA-4) prior to and/or during the co-culturing. In some embodiments,the population of PBMCs is obtained from the individual being treated.

In some embodiments, there is provided a method of treating a cancer inan individual comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs; (b) administering to the individual an effective amount of theactivated PBMCs; and (c) administering to the individual an effectiveamount of an inhibitor of CTLA-4. In some embodiments, the inhibitor ofCTLA-4 is an anti-CTLA-4 antibody, such as Ipilimumab. In someembodiments, the activated PBMCs and the inhibitor of CTLA-4 areadministered simultaneously, such as in the same composition. In someembodiments, the activated PBMCs and the inhibitor of CTLA-4 areadministered sequentially. In some embodiments, the PBMCs is contactedwith the plurality of tumor antigen peptides in the presence of acomposition that facilitates the uptake of the plurality of tumorantigen peptides by antigen presenting cells (such as dendritic cells)in the PBMCs. In some embodiments, the population of activated PBMCs isfurther contacted with IL-2. In some embodiments, the population ofPBMCs is contacted with the plurality of tumor antigen peptides in thepresence of an immune checkpoint inhibitor, such as an inhibitor ofCTLA-4. In some embodiments, the activated PBMCs are administered for atleast three times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated.

In some embodiments, the activated T cells (or the activated PBMCs) andthe immune checkpoint inhibitor are administered simultaneously. In someembodiments, the activated T cells (or the activated PBMCs) and theimmune checkpoint inhibitor are administered in a single composition. Insome embodiments, the immune checkpoint inhibitor is present in theco-culture. In some embodiments, the activated T cells (or the activatedPBMCs) and the immune checkpoint inhibitor are admixed prior to (such asimmediately prior to) the administration. In some embodiments, theactivated T cells (or the activated PBMCs) and the immune checkpointinhibitor are administered simultaneously via separate compositions.

In some embodiments, the activated T cells (or the activated PBMCs) andthe immune checkpoint inhibitor are administered sequentially. In someembodiments, the immune checkpoint inhibitor is administered prior tothe administration of the activated T cells (or the activated PBMCs). Insome embodiments, the immune checkpoint inhibitor is administered afterthe administration of the activated T cells (or the activated PBMCs).

Plurality of Tumor Antigen Peptides

All of the MASCT methods (including PBMC-based MASCT methods) and cellpreparation methods described herein use a plurality of tumor antigenpeptides (including neoantigen peptides) to prepare APCs (such asdendritic cells) and activated T cells, or activated PBMCs that cantrigger specific T cell response ex vivo and in vivo.

In some embodiments, each tumor antigen peptide in the MASCT methodcomprises about any of 1, 2, 3, 4, 5, or 10 epitopes from a singleprotein antigen (including a neoantigen). In some embodiments, eachtumor antigen peptide in the plurality of tumor antigen peptidescomprises at least one epitope recognizable by a T cell receptor. Insome embodiments, the plurality of tumor antigen peptides comprises atleast one tumor antigen peptide that comprises at least 2 epitopes froma single protein antigen. The tumor antigen peptide can be a naturallyderived peptide fragment from a protein antigen containing one or moreepitopes, or an artificially designed peptide with one or more naturalepitope sequences, wherein a linker peptide can optionally be placed inbetween adjacent epitope sequences. In some preferred embodiments, theepitopes contained in the same tumor antigen peptide are derived fromthe same protein antigen.

The tumor antigen peptide (including neoantigen peptide) may contain atleast one MHC-I epitope, at least one MHC-II epitope, or both MHC-Iepitope(s) and MHC-II epitope(s). In some embodiments, the plurality oftumor antigen peptides comprises at least one peptide comprising anMHC-I epitope. In some embodiments, the plurality of tumor antigenpeptides comprises at least one peptide comprising an MHC-II epitope. Insome embodiments, at least one tumor antigen peptide in the plurality oftumor antigen peptides comprises both MHC-I and MHC-II epitopes.

Special design strategies can be applied to the sequence of the tumorantigen peptides (including neoantigen peptides) in order to optimizethe immune response to dendritic cells loaded with the tumor antigenpeptides. Typically, a peptide longer than the exact epitope peptide canincrease uptake of the peptide into antigen presenting cells (such asdendritic cells). In some embodiments, an MHC-I or MHC-II epitopesequence is extended at the N terminus or the C terminus or both terminiaccording to the natural sequence of the protein harboring the epitopeto obtain an extended sequence, wherein the extended sequence isamenable for presentation by both class I and class II MHC molecules,and by different subtypes of MHC molecules in different individuals. Insome embodiments, the epitope sequence is extended at one or bothtermini by about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20amino acid residues to generate the extended epitope. In someembodiments, the peptides comprising an MHC-I or epitope furthercomprise additional amino acids flanking the epitope at the N-terminus,the C-terminus, or both. In some embodiments, each tumor antigen peptidein the plurality of tumor antigen peptides is about any of 10, 15, 20,25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids long.Different tumor antigen peptides in the plurality of tumor antigenpeptides may have the same length, or different lengths. In someembodiments, the plurality of tumor antigen peptides is each about 20-40amino acids long.

In some embodiments, the amino acid sequences of one or more epitopepeptides used to design a tumor antigen peptide in the presentapplication are based on sequences known in the art or available inpublic databases, such as the Peptide Database (van der Bruggen P et al.(2013) “Peptide database: T cell-defined tumor antigens. CancerImmunity. URL: www.cancerimmunity.org/peptide/). In some embodiments,the amino acid sequences of the one or more epitope peptides areselected from the group consisting of SEQ ID NOs: 1-35.

In some embodiments, the amino acid sequences of one or more epitopepeptides are predicted based on the sequence of the antigen protein(including neoantigens) using a bioinformatics tool for T cell epitopeprediction. Exemplary bioinformatics tools for T cell epitope predictionare known in the art, for example, see Yang X. and Yu X. (2009) “Anintroduction to epitope prediction methods and software” Rev. Med.Viral. 19(2): 77-96. In some embodiments, the sequence of the antigenprotein is known in the art or available in public databases. In someembodiments, the sequence of the antigen protein (including neoantigens)is determined by sequencing a sample (such as a tumor sample) of theindividual being treated.

The present invention contemplates tumor antigen peptides derived fromany tumor antigens and epitopes known in the art, including neoantigensand neoepitopes, or specially developed or predicted usingbioinformatics tools by the inventors.

In some embodiments, the plurality of tumor antigen peptides comprises afirst core group of general tumor antigen peptides. In some embodiments,the plurality of tumor antigen peptides further comprises a second groupof cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, neoantigen peptides are cancer-typespecific antigen peptides. In some embodiments, the plurality of tumorantigen peptides consists of the first core group of general tumorantigen peptides. In some embodiments, the plurality of tumor antigenpeptides consists of the first core group of general tumor antigenpeptides and the second group of cancer-type specific antigen peptides.In some embodiments, the plurality of tumor antigen peptides consists ofneoantigen peptides only. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and one or more neoantigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides, a second group of cancer-type specificantigen peptides, and one or more neoantigen peptides.

The first core group of general tumor antigen peptides is derived fromtumor antigens commonly expressed or overexpressed on the surface of avariety of cancers of different types. Therefore, the first core groupof general tumor antigen peptides is useful to prepare dendritic cells,or activated T cells used in any of the MASCT methods (includingPBMC-based MASCT methods), or in other treatment methods or cellpreparation methods described herein to treat individuals with differentcancer types. For example, in some embodiments, the first core group ofgeneral tumor antigen peptides is useful for methods described hereinfor treating a variety of cancers, such as lung cancer, colon cancer,gastric cancer, prostate cancer, melanoma, lymphoma, pancreatic cancer,ovarian cancer, breast cancer, glioma, esophageal cancer, nasopharyngealcarcinoma, cervical cancer, renal carcinoma, or hepatocellularcarcinoma. Exemplary tumor antigen peptides of the first core groupinclude, but are not limited to, peptides derived from hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, and CDCA1. Thefirst core group may comprise peptides derived from more than about anyof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 22, 25, 30, 40, or 50 tumor antigens. The first core group maycomprise about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 22, 25, 30, 40, or 50 general tumor antigenpeptides. In some embodiments, the first core group comprises more thanone general tumor antigen peptides. In some embodiments, the first coregroup comprises about 10 to about 20 general tumor antigen peptides. Insome embodiments, the first core group comprises general tumor antigenpeptides having more than one epitopes selected from the groupconsisting of SEQ ID NOs: 1-24.

The second group of cancer-type specific antigen peptides is derivedfrom tumor antigens that are expressed or overexpressed only in one or alimited number of cancer types. Therefore, the second group ofcancer-type specific antigen peptides is useful to prepare dendriticcells, activated T cells used in any of the MASCT methods, or in othertreatment methods or cell preparation methods described herein, to treatindividuals with a particular type of cancer. Exemplary cancer-typespecific antigen peptides for treating hepatocellular carcinoma (HCC)include, but are not limited to, peptides derived from AFP, and GPC3. Insome embodiments, one or more cancer-specific antigen peptide is avirus-specific antigen peptide derived from a virus that can inducecancer, or relates to cancer development in the individual wheninfecting the individual. In some embodiments, the virus-specificantigen peptide is specific to the subtype of the virus infecting theindividual. Exemplary virus-specific antigen peptides for treating anHCC patient with concurrent infection of HBV include, but are notlimited to, peptides derived from HBV core antigen, and HBV DNApolymerase. In some embodiments, the virus-specific antigen peptidescomprise at least one epitope selected from the group consisting of SEQID NOs: 31-35. In some embodiments, the second group comprisesvirus-specific antigen peptides derived from HBV antigens, wherein themethod is to treat hepatocellular carcinoma in an individual. In someembodiments, the second group comprises virus-specific antigen peptidesderived from HPV antigens, wherein the method is to treat cervicalcancer in an individual. In some embodiments, the second group comprisesvirus-specific antigen peptides derived from EBV antigens, wherein themethod is to treat nasopharyngeal carcinoma in an individual. The secondgroup of cancer-type specific antigen peptides may comprise peptidesderived from more than about any of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50cancer-type specific antigens. The second group of cancer-type specificantigen peptides may comprise more than about any of 0, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40,or 50 cancer-type specific antigen peptides. In some embodiments, thesecond group comprises more than one cancer-type specific antigenpeptides. In some embodiments, the second group comprises about 1 toabout 10 cancer-type specific antigen peptides. In some embodiments, thesecond group comprises cancer-type specific antigen peptides comprisingat least one epitope selected from the group consisting of SEQ ID NOs:25-35, wherein the cancer is hepatocellular carcinoma. In someembodiments, the type of cancer targeted by the cancer-type specificantigen peptides is selected from the group consisting essentially ofhepatocellular carcinoma, cervical cancer, nasopharyngeal carcinoma,breast cancer, and lymphoma.

In some embodiments, the plurality of tumor antigen peptides comprisesone or more (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)neoantigen peptides. The neoantigen peptides are derived fromneoantigens. Neoantigens are newly acquired and expressed antigenspresent in tumor cells of the individual, such as the individual beingtreated for cancer. In some embodiments, neoantigens are derived frommutant protein antigens that are only present in cancer cells, butabsent in normal cells. Neoantigens may be uniquely present in the tumorcells (such as all tumor cells or a portion of tumor cells) of theindividual being treated for cancer, or present in individuals havingsimilar types of cancer as the individual being treated. In someembodiments, the neoantigen is a clonal neoantigen. In some embodiments,the neoantigen is a subclonal neoantigen. In some embodiments, theneoantigen is present in at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or more tumor cells in the individual. In someembodiments, the neoantigen peptide comprises an MHC-I restrictedneoepitope. In some embodiments, the neoantigen peptide comprises anMHC-II restricted neoepitope. In some embodiments, the neoantigenpeptide is designed to facilitate presentation of the neoepitope by bothclass I and class II MHC molecules, for example, by extending theneoepitope at both the N- and the C-termini. Exemplary neoantigenpeptides include, but are not limited to, neoepitope derived from mutantKRAS (e.g., KRAS^(G12A)), PARP4 (e.g., PARP4^(T1170I)), MLL3 (e.g.MLL3^(C988F)) and MTHFR (e.g., MTHFR^(A222V)). In some embodiments, theneoantigen peptide comprises an epitope having a point mutation in asequence selected from the group consisting of SEQ ID Nos: 41-45. Insome embodiments, the neoantigen peptide comprises an epitope selectedfrom the group consisting of SEQ ID NOs: 36-40.

Neoantigen peptides can be selected based on the genetic profile of oneor more tumor sites of the individual being treated. In someembodiments, the genetic profile of the tumor sample comprises sequenceinformation of the full genome. In some embodiments, the genetic profileof the tumor sample comprises sequence information of the exome. In someembodiments, the genetic profile of the tumor sample comprises sequenceinformation of cancer-associated genes.

Neoantigen peptides suitable for use in the present invention may bederived from any mutant proteins, such as those encoded by mutantcancer-associated genes, in the tumor cells. In some embodiments, theneoantigen peptide comprises a single neoepitope derived from acancer-associated gene. In some embodiments, the neoantigen peptidecomprises more than one (such as 2, 3, or more) neoepitope derived froma cancer-associated gene. In some embodiments, the neoantigen peptidecomprises more than one (such as 2, 3, or more) neoepitope derived frommore than one (such as 2, 3, or more) cancer-associated genes. In someembodiments, the plurality of tumor antigens comprises a plurality ofneoantigen peptides derived from a single cancer-associated gene. Insome embodiments, the plurality of tumor antigens comprises a pluralityof neoantigen peptides derived from more than one (such as any of 2, 3,4, 5, or more) cancer-associated genes.

Cancer-associated genes are genes that are overexpressed or onlyexpressed in cancer cells, but not normal cells. Exemplarycancer-associated genes include, but are not limited to, ABL1, AKT1,AKT2, AKT3, ALK, ALOX12B, APC, AR, ARAF, ARID1A, ARID1B, ARID2, ASXL1,ATM, ATRX, AURKA, AURKB, AXL, B2M, BAP1, BCL2, BCL2L1, BCL2L12, BCL6,BCOR, BCORL1, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BUB1B,CADM2, CARD11, CBL, CBLB, CCND1, CCND2, CCND3, CCNE1, CD274, CD58,CD79B, CDCl73, CDH1, CDK1, CDK2, CDK4, CDK5, CDK6, CDK9, CDKN1A, CDKN1B,CDKN1C, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK2, CIITA, CREBBP, CRKL,CRLF2, CRTC1, CRTC2, CSF1R, CSF3R, CTNNB1, CUX1, CYLD, DDB2, DDR2,DEPDC5, DICER1, DIS3, DMD, DNMT3A, EED, EGFR, EP300, EPHA3, EPHA5,EPHA7, ERBB2, ERBB3, ERBB4, ERCC2, ERCC3, ERCC4, ERCC5, ESR1, ETV1,ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, FAM46C, FANCA, FANCC, FANCD2,FANCE, FANCF, FANCG, FAS, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FH, FKBP9,FLCN, FLT1, FLT3, FLT4, FUS, GATA3, GATA4, GATA6, GLI1, GLI2, GLI3,GNA11, GNAQ, GNAS, GNB2L1, GPC3, GSTM5, H3F3A, HNF1A, HRAS, ID3, IDH1,IDH2, IGF1R, IKZF1, IKZF3, INSIG1, JAK2, JAK3, KCNIP1, KDM5C, KDM6A,KDM6B, KDR, KEAP1, KIT, KRAS, LINC00894, LMO1, LMO2, LMO3, MAP2K1,MAP2K4, MAP3K1, MAPK1, MCL1, MDM2, MDM4, MECOM, MEF2B, MEN1, MET, MITF,MLH1, MLL (KMT2A), MLL2 (KTM2D), MPL, MSH2, MSH6, MTOR, MUTYH, MYB,MYBL1, MYC, MYCL1 (MYCL), MYCN, MYD88, NBN, NEGR1, NF1, NF2, NFE2L2,NFKBIA, NFKBIZ, NKX2-1, NOTCH1, NOTCH2, NPM1, NPRL2, NPRL3, NRAS, NTRK1,NTRK2, NTRK3, PALB2, PARK2, PAX5, PBRM1, PDCD1LG2, PDGFRA, PDGFRB, PHF6,PHOX2B, PIK3C2B, PIK3CA, PIK3R1, PIM1, PMS1, PMS2, PNRC1, PRAME, PRDM1,PRF1, PRKAR1A, PRKCI, PRKCZ, PRKDC, PRPF40B, PRPF8, PSMD13, PTCH1, PTEN,PTK2, PTPN11, PTPRD, QKI, RAD21, RAF1, RARA, RB1, RBL2, RECQL4, REL,RET, RFWD2, RHEB, RHPN2, ROS1, RPL26, RUNX1, SBDS, SDHA, SDHAF2, SDHB,SDHC, SDHD, SETBP1, SETD2, SF1, SF3B1, SH2B3, SLITRK6, SMAD2, SMAD4,SMARCA4, SMARCB1, SMC1A, SMC3, SMO, SOCS1, SOX2, SOX9, SQSTM1, SRC,SRSF2, STAG1, STAG2, STAT3, STATE, STK11, SUFU, SUZ12, SYK, TCF3,TCF7L1, TCF7L2, TERC, TERT, TET2, TLR4, TNFAIP3, TP53, TSC1, TSC2,U2AF1, VHL, WRN, WT1, XPA, XPC, XPO1, ZNF217, ZNF708, and ZRSR2.

In some embodiments, the plurality of tumor antigen peptides comprisesat least one (such as at least about any of 1, 5, 10, 15, 20, 25, 30,35, or 40) epitope selected from the group consisting of SEQ IDNOs:1-40. In some embodiments, the plurality of tumor antigen peptidescomprises at least one (such as at least about any of 1, 5, 10, 15, 20,or 24) epitope selected from the group consisting of SEQ ID NOs:1-24. Insome embodiments, the plurality of tumor antigen peptides comprises atleast one (such as about any of 1, 2, 3, 4, 5, or 6) epitope selectedfrom the group consisting of SEQ ID NOs:25-30. In some embodiments, theplurality of tumor antigen peptides comprises at least one (such asabout any of 1, 2, 3, 4, or 5) epitope selected from the groupconsisting of SEQ ID NOs:31-35. In some embodiments, the plurality oftumor antigen peptides comprises at least one (such as about any of 1,2, 3, 4, or 5) epitope selected from the group consisting of SEQ IDNOs:36-40 In some embodiments, the plurality of tumor antigen peptidescomprises at least one (such as at least about any of 1, 2, 3, 4, 5, 6,7, 8, 9, or 10) of the general tumor antigen peptides in FIG. 2B, or 2C.In some embodiments, the plurality of tumor antigen peptides comprisesat least one (such as at least about any of 1, 2, 3, 4, or 5) neoantigenpeptide in FIG. 29A. In some embodiments, the plurality of tumor antigenpeptides comprises at least one (such as at least about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) tumor antigen peptide inFIG. 2B, 2C, or 29A. In some embodiments, the plurality of tumor antigenpeptides comprises at least one (such as at least about any of 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more) tumor antigen peptideeach comprising one or more epitopes encoded by a cancer-associated geneselected from the group consisting of hTERT, p53, Survivin, NY-ESO-1,CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS,PARP4, MLL3, and MTHFR.

In some embodiments, the plurality of tumor antigen peptides comprisesat least 10 tumor antigen peptides. In some embodiments, each of the atleast 10 tumor antigen peptides comprises at least one epitope selectedfrom the group consisting of SEQ ID NOs: 1-40. In some embodiments, eachof the at least 10 tumor antigen peptides comprises at least one epitopeselected from the group consisting of SEQ ID NOs: 1-24. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides selected from the group consisting of thetumor antigen peptides in FIG. 2B. In some embodiments, the plurality oftumor antigen peptides comprises at least 10 tumor antigen peptidesselected from the group consisting of the tumor antigen peptides in 2C.In some embodiments, the plurality of tumor antigen peptides comprisesat least one neoantigen peptide in FIG. 29A.

In some embodiments, there is provided a composition comprising at least10 tumor antigen peptides, wherein each of the at least 10 tumor antigenpeptides comprises at least one epitope selected from the groupconsisting of SEQ ID NOs: 1-40. In some embodiments, there is provided acomposition comprising at least 10 tumor antigen peptides, wherein eachof the at least 10 tumor antigen peptides comprising at least oneepitope selected from the group consisting of SEQ ID NOs: 1-24. In someembodiments, there is provided a composition comprising at least 10tumor antigen peptides selected from the group consisting of the tumorantigen peptides in FIG. 2B. In some embodiments, there is provided acomposition comprising at least 10 tumor antigen peptides selected fromthe group consisting of the tumor antigen peptides in FIG. 2C and FIG.29A. In some embodiments, there is provided a composition comprising atleast 10 tumor antigen peptides each comprising an epitope encoded by acancer-associated gene selected from the group consisting of hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc,HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

In some embodiments, there is provided an isolated population ofdendritic cells loaded with a plurality of tumor antigen peptidesprepared by contacting a population of dendritic cells with a pluralityof tumor antigen peptides, wherein the plurality of tumor antigenpeptides comprises at least 10 tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides comprising a first core group of general tumorantigen peptides and optionally a second group of cancer-type specificantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises one or more neoantigen peptides. In some embodiments,each of the at least 10 tumor antigen peptides comprises at least oneepitope selected from the group consisting of SEQ ID NOs: 1-40. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides selected from the group consisting of thetumor antigen peptides in FIG. 2C and FIG. 29A. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides each comprising one or more epitopes encoded by acancer-associated gene selected from the group consisting of hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc,HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

In some embodiments, there is provided a method of preparing apopulation of activated T cells, comprising co-culturing a population ofT cells with a population of dendritic cells loaded with a plurality oftumor antigen peptides, wherein the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides. In some embodiments, thereis provided an isolated population of activated T cells prepared byco-culturing a population of T cells with a population of dendriticcells loaded with a plurality of tumor antigen peptides, wherein theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides comprising a first coregroup of general tumor antigen peptides and optionally a second group ofcancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, each of the at least 10 tumor antigenpeptides comprises at least one epitope selected from the groupconsisting of SEQ ID NOs: 1-40. In some embodiments, the plurality oftumor antigen peptides comprises at least 10 tumor antigen peptidesselected from the group consisting of the tumor antigen peptides in FIG.2C and FIG. 29A. In some embodiments, the plurality of tumor antigenpeptides comprises at least 10 tumor antigen peptides each comprisingone or more epitopes encoded by a cancer-associated gene selected fromthe group consisting of hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1, MET,RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, andMTHFR.

In some embodiments, there is provided a method of preparing apopulation of activated T cells, comprising: (a) inducingdifferentiation of a population of monocytes into a population ofdendritic cells; (b) contacting the population of dendritic cells with aplurality of tumor antigen peptides to obtain a population of dendriticcells loaded with the plurality of tumor antigen peptides; (c)co-culturing the population of dendritic cells loaded with the pluralityof tumor antigen peptides and a population of non-adherent PBMCs toobtain the population of activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs (such as from the individual), and wherein theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, there is provided an isolated populationof activated T cells prepared by (a) inducing differentiation of apopulation of monocytes into a population of dendritic cells; (b)contacting the population of dendritic cells with a plurality of tumorantigen peptides to obtain a population of dendritic cells loaded withthe plurality of tumor antigen peptides; (c) co-culturing the populationof dendritic cells loaded with the plurality of tumor antigen peptidesand a population of non-adherent PBMCs to obtain the population ofactivated T cells, wherein the population of monocytes and thepopulation of non-adherent PBMCs are obtained from a population of PBMCs(such as from the individual), and wherein the plurality of tumorantigen peptides comprises at least 10 tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides comprising a first core group of general tumorantigen peptides and optionally a second group of cancer-type specificantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises one or more neoantigen peptides. In some embodiments,each of the at least 10 tumor antigen peptides comprises at least oneepitope selected from the group consisting of SEQ ID NOs: 1-40. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides selected from the group consisting of thetumor antigen peptides in FIG. 2C and FIG. 29A. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides each comprising one or more epitopes encoded by acancer-associated gene selected from the group consisting of hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc,HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs, and administering to the individual an effective amount of theactivated PBMCs, wherein the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides. In some embodiments, thepopulation of PBMCs is contacted with the plurality of tumor antigenpeptides in the presence of an immune checkpoint inhibitor, such as aninhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3. Insome embodiments, the activated PBMCs are administered for at leastthree times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the population of PBMCsis obtained from the individual being treated. In some embodiments, themethod further comprises administering to the individual an effectiveamount of an immune checkpoint inhibitor, such as an inhibitor of PD-1,PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments,the plurality of tumor antigen peptides comprises at least 10 tumorantigen peptides comprising a first core group of general tumor antigenpeptides and optionally a second group of cancer-type specific antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises one or more neoantigen peptides. In some embodiments, each ofthe at least 10 tumor antigen peptides comprises at least one epitopeselected from the group consisting of SEQ ID NOs: 1-40. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides selected from the group consisting of thetumor antigen peptides in FIG. 2C and FIG. 29A. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides each comprising one or more epitopes encoded by acancer-associated gene selected from the group consisting of hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc,HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising optionally administering to the individual aneffective amount of dendritic cells loaded with a plurality of tumorantigen peptides, and administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing (such as in the presence of an immune checkpointinhibitor) a population of T cells with a population of dendritic cellsloaded with the plurality of tumor antigen peptides, wherein theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the population of T cells is contactedwith an immune checkpoint inhibitor (such as an inhibitor of PD-1,PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of dendritic cells loaded with the pluralityof tumor antigen peptides is prepared by contacting a population ofdendritic cells with the plurality of tumor antigen peptides. In someembodiments, the population of T cells and the population of dendriticcells are derived from the same individual. In some embodiments, thepopulation of T cells, the population of dendritic cells, the populationof PBMCs, or any combination thereof is derived from the individualbeing treated. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the plurality oftumor antigen peptides comprises at least 10 tumor antigen peptidescomprising a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises oneor more neoantigen peptides. In some embodiments, each of the at least10 tumor antigen peptides comprises at least one epitope selected fromthe group consisting of SEQ ID NOs: 1-40. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides selected from the group consisting of the tumor antigenpeptides in FIG. 2C and FIG. 29A. In some embodiments, the plurality oftumor antigen peptides comprises at least 10 tumor antigen peptides eachcomprising one or more epitopes encoded by a cancer-associated geneselected from the group consisting of hTERT, p53, Survivin, NY-ESO-1,CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS,PARP4, MLL3, and MTHFR.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells; (b) contacting thepopulation of dendritic cells with a plurality of tumor antigen peptidesto obtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and apopulation of non-adherent PBMCs to obtain the population of activated Tcells; and (e) administering to the individual an effective amount ofthe activated T cells, wherein the population of monocytes and thepopulation of non-adherent PBMCs are obtained from a population of PBMCs(such as from the individual), and wherein the plurality of tumorantigen peptides comprises at least 10 tumor antigen peptides. In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, orCTLA-4) prior to and/or during the co-culturing. In some embodiments,the method further comprises administering to the individual aneffective amount of an immune checkpoint inhibitor, such as an inhibitorof PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides comprising a first core group of general tumorantigen peptides and optionally a second group of cancer-type specificantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises one or more neoantigen peptides. In some embodiments,each of the at least 10 tumor antigen peptides comprises at least oneepitope selected from the group consisting of SEQ ID NOs: 1-40. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides selected from the group consisting of thetumor antigen peptides in FIG. 2C and FIG. 29A. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides each comprising one or more epitopes encoded by acancer-associated gene selected from the group consisting of hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc,HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

Precision MASCT

Further provided herein are precision MASCT methods that are customizedto the individual being treated based on the genetics and therapeuticresponse of the individual. Any of the MASCT methods described above maybe customized to provide a precision MASCT method.

The MASCT methods described herein in some embodiments are particularlysuitable for a certain population of individuals, such as individualswith a low mutation load (such as in the MHC genes) in the cancer (suchas all or a subset of cancer cells), and/or individuals with one or moreneoantigens.

Mutation Load

In some embodiments, the MASCT methods are particularly suitable for anindividual with a low total mutation load in the cancer of theindividual. In some embodiments, the MASCT methods are particularlysuitable for an individual with a low mutation load in thecancer-associated genes in the cancer of the individual. In someembodiments, the MASCT methods are particularly suitable for anindividual with a low mutation load in immune genes related to T cellresponse in the cancer of the individual. In some embodiments, the MASCTmethods are particularly suitable for an individual with a low mutationload in the MHC genes in the cancer of the individual. The mutation loadmay be mutation load in all cancer cells, or a subset of cancer cells,such as a primary or metastatic tumor site, for example, cells in atumor biopsy sample.

Thus, in some embodiments, there is provided a method of treating acancer in an individual, comprising: (a) optionally administering aneffective amount of dendritic cells loaded with a plurality of tumorantigen peptides; and (b) administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing a population of T cells with the population of dendriticcells loaded with the plurality of tumor antigen peptides, and whereinthe individual has a low mutation load in the cancer. In someembodiments, the interval between the administration of the dendriticcells and the administration of the activated T cells is about 7 days toabout 21 days (such as about 7 days to about 14 days, about 14 days toabout 21 days, about 10 days or about 14 days). In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, or about 14 days to about 21 days). In someembodiments, the population of T cell is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4)prior to and/or during the co-culturing. In some embodiments, thepopulation of dendritic cells and the population of T cells are derivedfrom the same individual, such as the individual being treated. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and optionally a second group of cancer-type specific antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises one or more neoantigen peptides. In some embodiments, themethod further comprises administering to the individual an effectiveamount of an immune checkpoint inhibitor, such as an inhibitor of PD-1,PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments,the mutation load of the cancer is determined by sequencing a tumorsample from the individual. In some embodiments, the individual has alow mutation load (such as no more than about 10 mutations, no mutationsin B2M, and/or no mutation in the functional regions) in one or more MEWgenes (such as MHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) selecting the individual for the methodbased on the mutation load in the cancer; (b) optionally administeringan effective amount of dendritic cells loaded with a plurality of tumorantigen peptides; and (c) administering to the individual an effectiveamount of activated T cells, wherein the activated T cells are preparedby co-culturing a population of T cells with the population of dendriticcells loaded with the plurality of tumor antigen peptides. In someembodiments, the interval between the administration of the dendriticcells and the administration of the activated T cells is about 7 days toabout 21 days (such as about 7 days to about 14 days, about 14 days toabout 21 days, about 10 days or about 14 days). In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, or about 14 days to about 21 days). In someembodiments, the population of T cell is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4)prior to and/or during the co-culturing. In some embodiments, thepopulation of dendritic cells and the population of T cells are derivedfrom the same individual, such as the individual being treated. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and optionally a second group of cancer-type specific antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises one or more neoantigen peptides. In some embodiments, themethod further comprises administering to the individual an effectiveamount of an immune checkpoint inhibitor, such as an inhibitor of PD-1,PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments,the mutation load of the cancer is determined by sequencing a tumorsample from the individual. In some embodiments, the individual has alow mutation load (such as no more than about 10 mutations, no mutationsin B2M, and/or no mutation in the functional regions) in one or more MEWgenes (such as MHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) optionally administering an effectiveamount of dendritic cells loaded with a plurality of tumor antigenpeptides; and (b) administering to the individual an effective amount ofactivated T cells, wherein the activated T cells are prepared byco-culturing a population of T cells with the population of dendriticcells loaded with the plurality of tumor antigen peptides, and whereinthe individual is selected for treatment based on having a low mutationload in the cancer. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the co-culturing is for about 7 daysto about 21 days (such as about 7 days to about 14 days, or about 14days to about 21 days). In some embodiments, the population of T cell iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the population of dendritic cells and the population of Tcells are derived from the same individual, such as the individual beingtreated. In some embodiments, the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides and optionally a second group ofcancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the mutation load ofthe cancer is determined by sequencing a tumor sample from theindividual. In some embodiments, the individual has a low mutation load(such as no more than about 10 mutations, no mutations in B2M, and/or nomutation in the functional regions) in one or more MEW genes (such asMHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells (such as in thepresence of GM-CSF and IL-4); (b) contacting the population of dendriticcells with a plurality of tumor antigen peptides (such as in thepresence of a plurality of Toll-like Receptor (TLR) agonists) to obtaina population of dendritic cells loaded with the plurality of tumorantigen peptides; (c) optionally administering to the individual aneffective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing (such as in the presence of aplurality of cytokines and optionally an anti-CD3 antibody) thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells; and (e) administering to the individual aneffective amount of the activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs (such as from the individual), and wherein theindividual has a low mutation load in the cancer. In some embodiments,the interval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, or about 14 days to about 21 days). In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, orCTLA-4) prior to and/or during the co-culturing. In some embodiments,the plurality of tumor antigen peptides comprises at least 10 tumorantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises a first core group of general tumor antigen peptidesand optionally a second group of cancer-type specific antigen peptides.In some embodiments, the plurality of tumor antigen peptides comprisesone or more neoantigen peptides. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, themutation load of the cancer is determined by sequencing a tumor samplefrom the individual. In some embodiments, the individual has a lowmutation load (such as no more than about 10 mutations, no mutations inB2M, and/or no mutation in the functional regions) in one or more MEWgenes (such as MHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) selecting the individual for the methodbased on the mutation load in the cancer; (b) inducing differentiationof a population of monocytes into a population of dendritic cells (suchas in the presence of GM-CSF and IL-4); (c) contacting the population ofdendritic cells with a plurality of tumor antigen peptides (such as inthe presence of a plurality of Toll-like Receptor (TLR) agonists) toobtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (d) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (e) co-culturing (such as in the presence of aplurality of cytokines and optionally an anti-CD3 antibody) thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells; and (f) administering to the individual aneffective amount of the activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs (such as from the individual). In some embodiments,the interval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, or about 14 days to about 21 days). In someembodiments, the population of non-adherent PBMCs is contacted with animmune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, orCTLA-4) prior to and/or during the co-culturing. In some embodiments,the plurality of tumor antigen peptides comprises at least 10 tumorantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises a first core group of general tumor antigen peptidesand optionally a second group of cancer-type specific antigen peptides.In some embodiments, the plurality of tumor antigen peptides comprisesone or more neoantigen peptides. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, themutation load of the cancer is determined by sequencing a tumor samplefrom the individual. In some embodiments, the individual has a lowmutation load (such as no more than about 10 mutations, no mutations inB2M, and/or no mutation in the functional regions) in one or more MEWgenes (such as MHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells (such as in thepresence of GM-CSF and IL-4); (b) contacting the population of dendriticcells with a plurality of tumor antigen peptides (such as in thepresence of a plurality of Toll-like Receptor (TLR) agonists) to obtaina population of dendritic cells loaded with the plurality of tumorantigen peptides; (c) optionally administering to the individual aneffective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing (such as in the presence of aplurality of cytokines and optionally an anti-CD3 antibody) thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells; and (e) administering to the individual aneffective amount of the activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs (such as from the individual), and wherein theindividual is selected for treatment based on having a low mutation loadin the cancer. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the co-culturing is for about 7 days toabout 21 days (such as about 7 days to about 14 days, or about 14 daysto about 21 days). In some embodiments, the population of non-adherentPBMCs is contacted with an immune checkpoint inhibitor (such as aninhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or during theco-culturing. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the plurality of tumorantigen peptides comprises at least 10 tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises a firstcore group of general tumor antigen peptides and optionally a secondgroup of cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the mutation load ofthe cancer is determined by sequencing a tumor sample from theindividual. In some embodiments, the individual has a low mutation load(such as no more than about 10 mutations, no mutations in B2M, and/or nomutation in the functional regions) in one or more MEW genes (such asMHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs (such as in the presence of an immune checkpoint inhibitor); and(b) administering to the individual an effective amount of the activatedPBMCs, wherein the individual has a low mutation load in the cancer. Insome embodiments, the activated PBMCs are administered for at leastthree times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises oneor more neoantigen peptides. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, themutation load of the cancer is determined by sequencing a tumor samplefrom the individual. In some embodiments, the individual has a lowmutation load (such as no more than about 10 mutations, no mutations inB2M, and/or no mutation in the functional regions) in one or more MEWgenes (such as MHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) selecting the individual for the methodbased on the mutation load in the cancer; (b) contacting a population ofPBMCs with a plurality of tumor antigen peptides to obtain a populationof activated PBMCs (such as in the presence of an immune checkpointinhibitor); and (c) administering to the individual an effective amountof the activated PBMCs. In some embodiments, the activated PBMCs areadministered for at least three times. In some embodiments, the intervalbetween each administration of the activated PBMCs is about 2 weeks toabout 5 months (such as about 3 months). In some embodiments, theactivated PBMCs are administered intravenously. In some embodiments, thepopulation of PBMCs is obtained from the individual being treated. Insome embodiments, the plurality of tumor antigen peptides comprises atleast 10 tumor antigen peptides. In some embodiments, the plurality oftumor antigen peptides comprises a first core group of general tumorantigen peptides and optionally a second group of cancer-type specificantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises one or more neoantigen peptides. In some embodiments,the method further comprises administering to the individual aneffective amount of an immune checkpoint inhibitor, such as an inhibitorof PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In someembodiments, the mutation load of the cancer is determined by sequencinga tumor sample from the individual. In some embodiments, the individualhas a low mutation load (such as no more than about 10 mutations, nomutations in B2M, and/or no mutation in the functional regions) in oneor more MEW genes (such as MHC-I genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides to obtain a population of activatedPBMCs (such as in the presence of an immune checkpoint inhibitor); and(b) administering to the individual an effective amount of the activatedPBMCs, wherein the individual is selected for treatment based on havinga low mutation load in the cancer. In some embodiments, the activatedPBMCs are administered for at least three times. In some embodiments,the interval between each administration of the activated PBMCs is about2 weeks to about 5 months (such as about 3 months). In some embodiments,the activated PBMCs are administered intravenously. In some embodiments,the population of PBMCs is obtained from the individual being treated.In some embodiments, the plurality of tumor antigen peptides comprisesat least 10 tumor antigen peptides. In some embodiments, the pluralityof tumor antigen peptides comprises a first core group of general tumorantigen peptides and optionally a second group of cancer-type specificantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises one or more neoantigen peptides. In some embodiments,the method further comprises administering to the individual aneffective amount of an immune checkpoint inhibitor, such as an inhibitorof PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In someembodiments, the mutation load of the cancer is determined by sequencinga tumor sample from the individual. In some embodiments, the individualhas a low mutation load (such as no more than about 10 mutations, nomutations in B2M, and/or no mutation in the functional regions) in oneor more MHC genes (such as MHC-I genes) in the cancer.

In some embodiments, a low mutation load of one or more genes is a lownumber of mutations accumulated on the one or more genes. In someembodiments, a total number of no more than about any of 500, 400, 300,200, 100, 50, 40, 30, 20, 10, 5 or fewer mutations indicate a lowmutation load. In some embodiments, no more than about any of 50, 40,30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1mutations in the one or more MHC genes indicate a low mutation load ofthe one or more MHC genes. In some embodiments, a low mutation load ofone or more genes is a low ratio between the number of mutationsaccumulated on the one or more genes (such as MHC genes) and the totalnumber of mutations in a selected set of genes (such ascancer-associated genes) or the full genome. In some embodiments, aratio of less than about any of 1:10, 1:15, 1:20, 1:25, 1:30, 1:40,1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200 or less between the number ofmutations in the one or more MHC genes and the total number of 333cancer-associated genes described in Example 5 indicate a low mutationload of the one or more MHC genes.

In some embodiments, the one or more MHC genes comprise MHC class Igenes (or loci). In some embodiments, the one or more MHC genes compriseMHC class II genes (or loci). In some embodiments, wherein theindividual is a human individual, the one or more MEW genes are selectedfrom the group consisting of HLA-A, HLA-B, HLA-C and B2M.

Exemplary mutations include, but are not limited to, deletion,frameshift, insertion, indel, missense mutation, nonsense mutation,point mutation, copy number variation, single nucleotide variation(SNV), silent mutation, splice site mutation, splice variant, genefusion, and translocation. In some embodiments, the copy numbervariation of the MHC gene is caused by structural rearrangement of thegenome, including deletions, duplications, inversion, and translocationof a chromosome or a fragment thereof. In some embodiments, themutations in the one or more MEW genes are selected from pointmutations, frameshift mutations, gene fusions, and copy numbervariations. In some embodiments, the mutations are in the protein-codingregion of the MEW genes. In some embodiments, the mutation is anonsynonymous mutation. In some embodiments, the mutation is not apolymorphism. In some embodiments, the mutation is present in normalcells of the individual. In some embodiments, the mutation is notpresent in normal cells of the individual. In some embodiments, themutation affects the physiochemical or functional properties, such asstability or binding affinity, of the MEW molecule encoded by theaffected gene. In some embodiments, the mutation results in anirreversible deficiency in the MEW molecule. In some embodiments, themutation reduces the binding affinity of the MEW molecule to T cellepitopes and/or T cell receptors. In some embodiments, the mutation is aloss-of-function mutation. In some embodiments, the mutation results inreversible deficiency in the MEW molecule. In some embodiments, themutation does not affect the binding affinity of the MEW molecule to Tcell epitopes and/or T cell receptors. In some embodiments, the mutationis a somatic mutation. In some embodiments, the mutation is a germlinemutation.

The mutations counted towards the mutation load may be present in allcancer cells or in a subset of cancer cells. In some embodiments, themutations are present in all cancer cells in the individual. In someembodiments, the mutations are present in all cancer cells of a tumorsite. In some embodiments, the mutations are clonal. In someembodiments, the mutations are subclonal. In some embodiments, themutations are present in at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or more cancer cells of the individual.

The mutations in certain MEW genes and/or in certain domains orpositions of the one or more MHC genes may have more profound influenceon the clinical response of the individual to the MASCT methodsdescribed herein. For example, loss-of-function mutations may occur inthe leader peptide sequence, a3 domain (which binds the CD8 co-receptorof T cells), a1 peptide binding domain, or a2 peptide binding domain ofthe HLA molecule; see, for example, Shukla S. et al. NatureBiotechnology 33, 1152-1158 (2015), incorporated herein by reference.Mutations in B2M (β2-macroglobulin) gene may also promote tumor escapephenotypes. See, for example, Monica B et al. Cancer Immunol. Immu.,(2012) 61: 1359-1371. In some embodiments, presence of any number (suchas 1, 2, 3, 4, 5, or more) of mutations in the functional regions of theone or more MHC genes, such as the leader peptide sequence, a1 domain,a2 domain, or a3 domain, indicates a high mutation load. In someembodiments, presence of any number (such as 1, 2, 3, 4, 5, or more)loss-of-function mutations in the one or more MHC genes (such as HLA-A,HLA-B or HLA-C genes in human individuals) indicates a high mutationload. In some embodiments, a low mutation load in the one or more MHCgenes comprises no mutation in the functional regions, including leaderpeptide sequence, a1 domain (for example, residues in direct contactwith the CD8 co-receptor), a2 domain, and a3 domain (for example,residues in direct contact with the epitope), of the one or more MHCgenes (such as HLA-A, HLA-B or HLA-C genes). In some embodiments,presence of any number of mutations (such as loss-of-function mutations)in the B2M gene indicates a high mutation load. In some embodiments, alow mutation load in the one or more MHC genes comprises no mutation inthe B2M gene.

The mutation load of one or more genes (such as MHC genes) may bedetermined by any known methods in the art, including, but not limitedto, genomic DNA sequencing, exome sequencing, or other DNAsequencing-based methods using Sanger sequencing or next generationsequencing platforms; polymerase chain reaction assays; in situhybridization assays; and DNA microarrays.

In some embodiments, the mutation load of the one or more MHC genes isdetermined by sequencing a tumor sample from the individual. In someembodiments, the sequencing is next generation sequencing. In someembodiments, the sequencing is full genome sequencing. In someembodiments, the sequencing is exome sequencing. In some embodiments,the sequencing is targeted sequencing of candidate genes, such ascancer-associated genes plus HLA genes. For example, ONCOGXONE™ Plus(Admera Health), are available to sequence cancer-associated genes andHLA loci with high sequencing depth. In some embodiments, the samesequencing data can be used to determine the mutation load of the one ormore MEW genes and to identify neoantigens in the individual.

In some embodiments, the tumor sample is a tissue sample. In someembodiments, the tumor sample is a tumor biopsy sample, such as fineneedle aspiration of tumor cells or laparoscopy obtained tumor cells(such as including tumor stroma). In some embodiments, the tumor sampleis freshly obtained. In some embodiments, the tumor sample is frozen. Insome embodiments, the tumor sample is a Formaldehyde Fixed-ParaffinEmbedded (FFPE) sample. In some embodiments, the tumor sample is a cellsample. In some embodiments, the tumor sample comprises a circulatingmetastatic cancer cell. In some embodiments, the tumor sample isobtained by sorting circulating tumor cells (CTCs) from blood. In someembodiments, nucleic acids (such as DNA and/or RNA) are extracted fromthe tumor sample for the sequencing analysis. In some embodiments, thesequencing data of the tumor sample is compared to the sequencing dataof a reference sample, such as a sample of a healthy tissue from thesame individual, or a sample of a healthy individual, to identifymutations and determine mutation load in the tumor cells. In someembodiments, the sequencing data of the tumor sample is compared to thereference sequences from a genome database to identify mutations anddetermine mutation load in the tumor cells.

Neoantigen Peptides

In some embodiments, the MASCT methods described herein are particularlysuitable for treating an individual with one or more neoantigens. Any ofthe MASCT methods described herein using one or more neoantigen peptidesin the plurality of tumor antigen peptides may further comprise thesteps of selecting the individual for the method of treating based onhaving one or more (such as at least 5) neoantigens in the individual,and/or the steps of: (i) identifying a neoantigen of the individual; and(ii) incorporating a neoantigen peptide derived from the neoantigen inthe plurality of tumor antigen peptides for use in the MASCT method.

Thus, in some embodiments, there is provided a method of treating acancer in an individual, comprising: (a) identifying a neoantigen of theindividual; (b) incorporating a neoantigen peptide in a plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen; (c) preparing a population of dendriticcells loaded with the plurality of tumor antigen peptides; (d)optionally administering an effective amount of dendritic cells loadedwith the plurality of tumor antigen peptides; (e) co-culturing apopulation of T cells with the population of dendritic cells loaded withthe plurality of tumor antigen peptides; and (f) administering to theindividual an effective amount of activated T cells, wherein theindividual has one or more neoantigens. In some embodiments, theinterval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the co-culturingis for about 7 days to about 21 days (such as about 7 days to about 14days, or about 14 days to about 21 days). In some embodiments, thepopulation of T cell is contacted with an immune checkpoint inhibitor(such as an inhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or duringthe co-culturing. In some embodiments, the dendritic cells loaded withthe plurality of tumor antigen peptides are administered subcutaneously.In some embodiments, the dendritic cells loaded with the plurality oftumor antigen peptides are administered for at least three times. Insome embodiments, the activated T cells are administered intravenously.In some embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the population of dendritic cells andthe population of T cells are derived from the same individual, such asthe individual being treated. In some embodiments, the plurality oftumor antigen peptides comprises at least 10 tumor antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises afirst core group of general tumor antigen peptides and optionally asecond group of cancer-type specific antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises aplurality of neoantigen peptides. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofan immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MEW genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) selecting the individual for the methodof treating based having one or more (such as at least 5) neoantigens inthe individual; (b) identifying a neoantigen of the individual; (c)incorporating a neoantigen peptide in a plurality of tumor antigenpeptides, wherein the neoantigen peptide comprises a neoepitope in theneoantigen; (d) preparing a population of dendritic cells loaded withthe plurality of tumor antigen peptides; (e) optionally administering aneffective amount of dendritic cells loaded with the plurality of tumorantigen peptides; (f) co-culturing a population of T cells with thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides; and (g) administering to the individual an effective amount ofactivated T cells. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the co-culturing is for about 7 days toabout 21 days (such as about 7 days to about 14 days, or about 14 daysto about 21 days). In some embodiments, the population of T cell iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, theactivated T cells are administered intravenously. In some embodiments,the activated T cells are administered for at least three times. In someembodiments, the population of dendritic cells and the population of Tcells are derived from the same individual, such as the individual beingtreated. In some embodiments, the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides and optionally a second group ofcancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a plurality of neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the individual isselected for the method of treating based on having a low mutation load(such as in one or more MEW genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) identifying a neoantigen of theindividual; (b) incorporating a neoantigen peptide in a plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen; (c) preparing a population of dendriticcells loaded with the plurality of tumor antigen peptides; (d)optionally administering an effective amount of dendritic cells loadedwith the plurality of tumor antigen peptides; (e) co-culturing apopulation of T cells with the population of dendritic cells loaded withthe plurality of tumor antigen peptides; and (f) administering to theindividual an effective amount of activated T cells, wherein theindividual is selected for the method of treating based on having one ormore (such as at least 5) neoantigens. In some embodiments, the intervalbetween the administration of the dendritic cells and the administrationof the activated T cells is about 7 days to about 21 days (such as about7 days to about 14 days, about 14 days to about 21 days, about 10 daysor about 14 days). In some embodiments, the co-culturing is for about 7days to about 21 days (such as about 7 days to about 14 days, or about14 days to about 21 days). In some embodiments, the population of T cellis contacted with an immune checkpoint inhibitor (such as an inhibitorof PD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, theactivated T cells are administered intravenously. In some embodiments,the activated T cells are administered for at least three times. In someembodiments, the population of dendritic cells and the population of Tcells are derived from the same individual, such as the individual beingtreated. In some embodiments, the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides and optionally a second group ofcancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a plurality of neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the individual isselected for the method of treating based on having a low mutation load(such as in one or more MEW genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) identifying a neoantigen of theindividual; (b) incorporating a neoantigen peptide in a plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen; (c) inducing differentiation of apopulation of monocytes into a population of dendritic cells (such as inthe presence of GM-CSF and IL-4); (d) contacting the population ofdendritic cells with a plurality of tumor antigen peptides (such as inthe presence of a plurality of Toll-like Receptor (TLR) agonists) toobtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (e) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (f) co-culturing (such as in the presence of aplurality of cytokines and optionally an anti-CD3 antibody) thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells; and (g) administering to the individual aneffective amount of the activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs (such as from the individual), and wherein theindividual has one or more neoantigens. In some embodiments, theinterval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the co-culturingis for about 7 days to about 21 days (such as about 7 days to about 14days, or about 14 days to about 21 days). In some embodiments, thepopulation of non-adherent PBMCs is contacted with an immune checkpointinhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) prior toand/or during the co-culturing. In some embodiments, the activated Tcells are administered intravenously. In some embodiments, the activatedT cells are administered for at least three times. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered subcutaneously. In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered for at least three times. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises aplurality of neoantigen peptides of the individual. In some embodiments,the method further comprises administering to the individual aneffective amount of an immune checkpoint inhibitor, such as an inhibitorof PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In someembodiments, the individual is selected for the method of treating basedon having a low mutation load (such as in one or more MHC genes) in thecancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) selecting the individual for the methodof treating based on having one or more (such as at least 5) neoantigensin the individual; (b) identifying a neoantigen of the individual; (c)incorporating a neoantigen peptide in a plurality of tumor antigenpeptides, wherein the neoantigen peptide comprises a neoepitope in theneoantigen; (d) inducing differentiation of a population of monocytesinto a population of dendritic cells (such as in the presence of GM-CSFand IL-4); (e) contacting the population of dendritic cells with aplurality of tumor antigen peptides (such as in the presence of aplurality of Toll-like Receptor (TLR) agonists) to obtain a populationof dendritic cells loaded with the plurality of tumor antigen peptides;(f) optionally administering to the individual an effective amount ofthe dendritic cells loaded with the plurality of tumor antigen peptides;(g) co-culturing (such as in the presence of a plurality of cytokinesand optionally an anti-CD3 antibody) the population of dendritic cellsloaded with the plurality of tumor antigen peptides and a population ofnon-adherent PBMCs to obtain the population of activated T cells; and(h) administering to the individual an effective amount of the activatedT cells, wherein the population of monocytes and the population ofnon-adherent PBMCs are obtained from a population of PBMCs (such as fromthe individual). In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the co-culturing is for about 7 days toabout 21 days (such as about 7 days to about 14 days, or about 14 daysto about 21 days). In some embodiments, the population of non-adherentPBMCs is contacted with an immune checkpoint inhibitor (such as aninhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or during theco-culturing. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the plurality of tumorantigen peptides comprises at least 10 tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises a firstcore group of general tumor antigen peptides and optionally a secondgroup of cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a plurality neoantigenpeptides of the individual. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MEW genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) identifying a neoantigen of theindividual; (b) incorporating a neoantigen peptide in a plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen; (c) inducing differentiation of apopulation of monocytes into a population of dendritic cells (such as inthe presence of GM-CSF and IL-4); (d) contacting the population ofdendritic cells with a plurality of tumor antigen peptides (such as inthe presence of a plurality of Toll-like Receptor (TLR) agonists) toobtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (e) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (f) co-culturing (such as in the presence of aplurality of cytokines and optionally an anti-CD3 antibody) thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells; and (g) administering to the individual aneffective amount of the activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs (such as from the individual), and wherein theindividual is selected for the method of treating based on having one ormore (such as at least 5) neoantigens. In some embodiments, the intervalbetween the administration of the dendritic cells and the administrationof the activated T cells is about 7 days to about 21 days (such as about7 days to about 14 days, about 14 days to about 21 days, about 10 daysor about 14 days). In some embodiments, the co-culturing is for about 7days to about 21 days (such as about 7 days to about 14 days, or about14 days to about 21 days). In some embodiments, the population ofnon-adherent PBMCs is contacted with an immune checkpoint inhibitor(such as an inhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or duringthe co-culturing. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the plurality of tumorantigen peptides comprises at least 10 tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises a firstcore group of general tumor antigen peptides and optionally a secondgroup of cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a plurality neoantigenpeptides of the individual. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MEW genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) identifying a neoantigen of theindividual; (b) incorporating a neoantigen peptide in a plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen; (c) contacting a population of PBMCs withthe plurality of tumor antigen peptides to obtain a population ofactivated PBMCs (such as in the presence of an immune checkpointinhibitor); and (d) administering to the individual an effective amountof the activated PBMCs, wherein the individual has one or moreneoantigens. In some embodiments, the activated PBMCs are administeredfor at least three times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises aplurality of neoantigen peptides. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofan immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MEW genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) selecting the individual for the methodof treating based on having one or more (such as at least 5) neoantigensin the individual; (b) identifying a neoantigen of the individual; (c)incorporating a neoantigen peptide in a plurality of tumor antigenpeptides, wherein the neoantigen peptide comprises a neoepitope in theneoantigen; (d) contacting a population of PBMCs with the plurality oftumor antigen peptides to obtain a population of activated PBMCs (suchas in the presence of an immune checkpoint inhibitor); and (e)administering to the individual an effective amount of the activatedPBMCs. In some embodiments, the activated PBMCs are administered for atleast three times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises aplurality of neoantigen peptides. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofan immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MEW genes) in the cancer.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) identifying a neoantigen of theindividual; (b) incorporating a neoantigen peptide in a plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen; (c) contacting a population of PBMCs withthe plurality of tumor antigen peptides to obtain a population ofactivated PBMCs (such as in the presence of an immune checkpointinhibitor); and (d) administering to the individual an effective amountof the activated PBMCs, wherein the individual is selected for themethod of treating based on having one or more (such as at least 5)neoantigens. In some embodiments, the activated PBMCs are administeredfor at least three times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises aplurality of neoantigen peptides. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofan immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MHC genes) in the cancer.

The individual may have any number (such as any of at least 1, 2, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 100 or more) of neoantigensin order to benefit from the MASCT method using a plurality of tumorantigen peptides comprising a neoantigen peptide. In some embodiments,the MASCT method is particularly suitable for an individual having atleast about any of 4, 5, 6, 7, 8, 10, 15, 20, 50, 100 or moreneoantigens. In some embodiments, the neoantigen comprises one or moreneoepitopes. In some embodiments, the MASCT method is particularlysuitable for an individual having at least about any of 4, 5, 6, 7, 8,10, 15, 20, 50, 100 or more neoepitopes. In some embodiments, the T cellepitopes are MHC-I restricted epitopes. In some embodiments, theneoepitope has a higher affinity to the MHC molecules of the individualthan the corresponding wildtype T cell epitope. In some embodiments, theneoepitope has higher affinity to a model T cell receptor than thecorresponding wildtype T cell epitope. In some embodiments, theneoantigen (or neoepitope) is a clonal neoantigen. In some embodiments,the neoantigen (or neoepitope) is a subclonal neoantigen. In someembodiments, the neoantigen (or neoepitope) is present in at least aboutany of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more tumorcells in the individual.

The number of neoantigens may be combined with other biomarkers orselection criteria to select an individual for any of the MASCT methodsdescribed herein. In some embodiments, the MASCT method is particularlysuitable for an individual having a low mutation load (such as in one ormore MHC genes) in the cancer cells, and at least about any of 4, 5, 6,7, 8, 10 or more neoantigens (such as neoantigens with high affinityMHC-I restricted neoepitopes).

In some embodiments, there is provided a method of providing a prognosisfor the individual based on the mutation load in the cancer of theindividual, and/or the number of neoantigens in the individual, whereinthe prognosis predicts the clinical response of the individual to any ofthe MASCT methods described herein. In some embodiments, the individualis categorized based on the prognosis into one of the following threecategories: (1) benefit from MHC-restricted intervention (such as MASCTtreatment); (2) potential benefit from MHC-restricted intervention (suchas MASCT treatment); and (3) no benefit from MHC-restricted intervention(such as MASCT treatment). In some embodiments, an individual ispredicted to benefit from MHC-restricted intervention (such as MASCTtreatment) if the individual has no mutation in B2M gene, no mutation inthe functional regions (such as leader peptide sequence, a1 domain, a2domain or a3 domain) of MHC genes, no more than 2 mutations in an MHC-Igene (such as HLA-I A, B, and/or C gene), and/or more than 5 mutations.In some embodiments, an individual is predicted to potentially benefitfrom MHC-restricted intervention (such as MASCT treatment) if theindividual has no mutation in B2M gene, no mutation in the functionalregions (such as leader peptide sequence, a1 domain, a2 domain or a3domain) of MHC genes, no more than about 10 mutations in MHC-I genes(such as HLA-I A, B, and/or C gene), and/or no more than 5 mutations. Insome embodiments, an individual is predicted to have no benefit fromMHC-restricted intervention (such as MASCT treatment) if the individualhas a mutation in B2M, or have a high mutation load (such as at least 10mutations) in the MHC genes (such as MHC-I genes). In some embodiments,the individual is selected for the MASCT method if the individual ispredicted to benefit or potentially benefit from MHC-restrictedintervention (such as MASCT treatment).

Any number (such as any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) ofneoantigen peptides may be designed based on the neoantigens of theindividual and to be incorporated in the plurality of tumor antigenpeptides for use in any of the MASCT methods described herein. In someembodiments, the plurality of tumor antigen peptides comprises a singleneoantigen peptide. In some embodiments, the plurality of tumor antigenpeptides comprises a plurality of neoantigen peptides. Each neoantigenpeptide may comprise one or more neoepitopes from a neoantigen of theindividual. In some embodiments, the neoepitope is a T cell epitope.Methods of designing a neoantigen peptide based on a neoantigen aredescribed in the section “Plurality of tumor antigen peptides.”

The neoantigens in the individual may be identified using any knownmethods in the art. In some embodiments, the neoantigen is identifiedbased on the genetic profile of a tumor sample from the individual. Eachneoantigen comprises one or more neoepitopes. In some embodiments, theone or more neoepitopes in the neoantigen are identified based on thegenetic profile of the tumor sample. Any known genetic profilingmethods, such as next generation sequencing (NGS) methods, microarrays,or proteomic methods may be used to provide the genetic profile of thetumor sample.

In some embodiments, the neoantigen is identified by sequencing a tumorsample from the individual. In some embodiments, the sequencing is nextgeneration sequencing. In some embodiments, the sequencing isfull-genome sequencing. In some embodiments, the sequencing is exomesequencing. In some embodiments, the sequencing is targeted sequencingof candidate genes, such as cancer-associated genes. Many commercial NGScancer panels, for example, ONCOGXONE™ Plus (Admera Health), areavailable to sequence cancer-associated genes with high sequencingdepth.

In some embodiments, the tumor sample is a tissue sample. In someembodiments, the tumor sample is a tumor biopsy sample, such as fineneedle aspiration of tumor cells or laparoscopy obtained tumor cells(such as including tumor stroma). In some embodiments, the tumor sampleis freshly obtained. In some embodiments, the tumor sample is frozen. Insome embodiments, the tumor sample is a Formaldehyde Fixed-ParaffinEmbedded (FFPE) sample. In some embodiments, the tumor sample is a cellsample. In some embodiments, the tumor sample comprises a circulatingmetastatic cancer cell. In some embodiments, the tumor sample isobtained by sorting circulating tumor cells (CTCs) from blood. In someembodiments, nucleic acids (such as DNA and/or RNA) are extracted fromthe tumor sample for the sequencing analysis. In some embodiments,proteins are extracted from the tumor sample for the sequencinganalysis.

In some embodiments, the genetic profile of the tumor sample is comparedto the genetic profile of a reference sample, such as a sample of ahealthy tissue from the same individual, or a sample of a healthyindividual, to identify candidate mutant genes in the tumor cells. Insome embodiments, the genetic profile of the tumor sample is compared tothe reference sequences from a genome database to identify candidatemutant genes in the tumor cells. In some embodiments, the candidatemutant genes are cancer-associated genes. In some embodiments, eachcandidate mutant gene comprises one or more mutations, such asnon-synonymous substitutions, indel (insertion or deletion), or genefusion, which may give rise to a neoantigen. Common Single NucleotidePolymorphisms (SNPs) are excluded from the candidate mutations.

In some embodiments, neoepitopes in neoantigens are identified from thecandidate mutant proteins. In some embodiments, the neoepitopes arepredicted in silico. Exemplary bioinformatics tools for T cell epitopeprediction are known in the art, for example, see Yang X. and Yu X.(2009) “An introduction to epitope prediction methods and software” Rev.Med. Virol. 19(2): 77-96. Factors considered in the T cell epitopeprediction algorithms include, but are not limited to, MHC subtype ofthe individual, sequence-derived physiochemical properties of the T cellepitope, MHC binding motifs, proteasomal cleavage pattern, transporterassociated with antigen processing (TAP) transport efficiency, MHCbinding affinity, peptide-MHC stability, and T-cell receptor bindingaffinity. In some embodiments, the neoepitope is an MHC-I restrictedepitope. In some embodiments, the neoepitope is an MHC-II restrictedepitope.

In some embodiments, the neoepitope has high affinity to the MHCmolecules of the individual. In some embodiments, the method furthercomprises determining the MHC subtype of the individual, for example,from the sequencing data, to identify one or more MHC molecules of theindividual. In some embodiments, the method further comprisesdetermining the affinity of the neoepitope to an MHC molecule, such asan MHC class I molecule. In some embodiments, the method comprisesdetermining the affinity of the neoepitope to one or more MHC (such asMHC class I) molecules of the individual. In some embodiments, theaffinity of the neoepitope to one or more MHC molecules of theindividual is compared to the affinity of the corresponding wildtypeepitope to the one or more MHC molecules of the individual. In someembodiments, the neoepitope is selected for having a higher (such as atleast about any of 1.5, 2, 5, 10, 15, 20, 25, 50, 100, or more times)affinity to the one or more MHC molecules (such as MHC-I molecules) ofthe individual than the corresponding wildtype epitope. In someembodiments, the MHC binding affinity is predicted in silico using anyknown tools or methods in the art. In some embodiments, the MHC bindingaffinity is determined experimentally, such as using an in vitro bindingassay.

In some embodiments, the method further comprises determining theaffinity of the complex comprising the neoepitope and an MHC molecule(such as an MHC class I molecule of the individual) to a T cellreceptor. In some embodiments, the affinity of the complex comprisingthe neoepitope and the MHC molecule to the T cell receptor is comparedto that of the complex comprising the corresponding wildtype epitope andthe MHC molecule. In some embodiments, the MHC molecule is from theindividual. In some embodiments, the T cell receptor is on the surfaceof one or more T cells of the individual. In some embodiments, theneoepitope is selected for having a higher (such as at least about anyof 1.5, 2, 5, 10, 15, 20, 25, 50, 100, or more times) affinity in acomplex comprising the neoepitope and an MHC molecule to a T cellreceptor model than the corresponding wildtype epitope. In someembodiments, the TCR binding affinity is predicted in silico using anyknown tools or methods in the art. In some embodiments, the TCR bindingaffinity is determined experimentally, for example, by determining the Tcell response against the neoepitope.

In some embodiments, the neoantigen (or the neoepitope) is identifiedfurther based on the expression level of the neoantigen (or theneoepitope) in the tumor sample. Expression level of the neoantigen (orthe neoepitope) may be determined using any methods for quantificationof mRNA or protein levels known in the art, such as RT-PCR,antibody-based assays, mass spectrometry. In some embodiments, theexpression level of the neoantigen (or the neoepitope) is determinedfrom the sequencing data of the tumor sample. In some embodiments, theneoantigen (or the neoepitope) is expressed in the tumor cells at alevel of at least about any of 10, 20, 50, 100, 200, 500, 1000, 2000,5000, 10⁴, or more copies per cell. In some embodiments, the neoantigen(or the neoepitope) is expressed at a level of more than about any of1.5, 2, 5, 10, 20, 50, 100, or more times than the correspondingwildtype protein (or the corresponding wildtype epitope) in the tumorcells.

In some embodiments, the neoantigen peptide is selected or identified bythe steps comprising: (a) sequencing a tumor sample from the individualto identify a neoantigen; (b) identifying a neoepitope in theneoantigen; optionally (c) determining the MHC subtype of the individual(e.g., using the sequencing data) to identify an MHC molecule of theindividual; optionally (d) determining the affinity of the neoepitope tothe MHC molecule of the individual; optionally (e) determining theaffinity of the complex comprising the neoepitope and the MHC moleculeto a T cell receptor; and (f) obtaining a peptide comprising theneoepitope to provide the neoantigen peptide. In some embodiments, theneoepitope has higher affinity to the MHC molecule (such as MHC-Imolecule) of the individual and/or higher affinity in the complexcomprising the neoepitope and the MHC molecule to the TCR as compared tothe complex comprising the corresponding wildtype T cell epitope and theMHC molecule. In some embodiments, the neoepitope is extended at the Nterminus or the C terminus or both termini according to the naturalsequence of the neoantigen harboring the epitope to obtain an extendedsequence, wherein the extended sequence is amenable for presentation byboth class I and class II MHC molecules. Any of the MASCT methodsdescribed herein using one or more neoantigen peptides may furthercomprise any one or more of the neoantigen selection/identificationsteps.

Thus, in some embodiments, there is provided a method of treating acancer in an individual, comprising: (a) sequencing a tumor sample fromthe individual to identify a neoantigen; (b) identifying a neoepitope inthe neoantigen; optionally (c) determining the MHC subtype of theindividual (e.g., using the sequencing data) to identify an MHC moleculeof the individual; optionally (d) determining the affinity of theneoepitope to the MHC molecule of the individual; optionally (e)determining the affinity of the complex comprising the neoepitope andthe MHC molecule to a T cell receptor; (f) incorporating a neoantigenpeptide comprising the neoepitope in a plurality of tumor antigenpeptides; (g) preparing a population of dendritic cells loaded with theplurality of tumor antigen peptides; (h) optionally administering aneffective amount of dendritic cells loaded with the plurality of tumorantigen peptides; (i) co-culturing a population of T cells with thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides; and (j) administering to the individual an effective amount ofactivated T cells. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the co-culturing is for about 7 days toabout 21 days (such as about 7 days to about 14 days, or about 14 daysto about 21 days). In some embodiments, the population of T cell iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the population of dendritic cells and the population of Tcells are derived from the same individual, such as the individual beingtreated. In some embodiments, the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides and optionally a second group ofcancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a plurality of neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the individual isselected for the method of treating based on having a low mutation load(such as in one or more MHC genes) in the cancer. In some embodiments,the method further comprises selecting the individual for the method oftreating based on having one or more (such as at least 5) neoantigens inthe individual.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) sequencing a tumor sample from theindividual to identify a neoantigen; (b) identifying a neoepitope in theneoantigen; optionally (c) determining the MHC subtype of the individual(e.g., using the sequencing data) to identify an MHC molecule of theindividual; optionally (d) determining the affinity of the neoepitope tothe MHC molecule of the individual; optionally (e) determining theaffinity of the complex comprising the neoepitope and the MHC moleculeto a T cell receptor; (f) incorporating a neoantigen peptide comprisingthe neoepitope in a plurality of tumor antigen peptides; (g) inducingdifferentiation of a population of monocytes into a population ofdendritic cells (such as in the presence of GM-CSF and IL-4); (h)contacting the population of dendritic cells with a plurality of tumorantigen peptides (such as in the presence of a plurality of Toll-likeReceptor (TLR) agonists) to obtain a population of dendritic cellsloaded with the plurality of tumor antigen peptides; (i) optionallyadministering to the individual an effective amount of the dendriticcells loaded with the plurality of tumor antigen peptides; (j)co-culturing (such as in the presence of a plurality of cytokines andoptionally an anti-CD3 antibody) the population of dendritic cellsloaded with the plurality of tumor antigen peptides and a population ofnon-adherent PBMCs to obtain the population of activated T cells; and(k) administering to the individual an effective amount of the activatedT cells, wherein the population of monocytes and the population ofnon-adherent PBMCs are obtained from a population of PBMCs (such as fromthe individual), and wherein the individual has one or more neoantigens.In some embodiments, the interval between the administration of thedendritic cells and the administration of the activated T cells is about7 days to about 21 days (such as about 7 days to about 14 days, about 14days to about 21 days, about 10 days or about 14 days). In someembodiments, the co-culturing is for about 7 days to about 21 days (suchas about 7 days to about 14 days, or about 14 days to about 21 days). Insome embodiments, the population of non-adherent PBMCs is contacted withan immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, orCTLA-4) prior to and/or during the co-culturing. In some embodiments,the activated T cells are administered intravenously. In someembodiments, the activated T cells are administered for at least threetimes. In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and optionally a second group of cancer-type specific antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a plurality neoantigen peptides of the individual. In someembodiments, the method further comprises administering to theindividual an effective amount of an immune checkpoint inhibitor, suchas an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, orLAG-3. In some embodiments, the individual is selected for the method oftreating based on having a low mutation load (such as in one or more MHCgenes) in the cancer. In some embodiments, the method further comprisesselecting the individual for the method of treating based on having oneor more (such as at least 5) neoantigens in the individual.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) sequencing a tumor sample from theindividual to identify a neoantigen; (b) identifying a neoepitope in theneoantigen; optionally (c) determining the MHC subtype of the individual(e.g., using the sequencing data) to identify an MHC molecule of theindividual; optionally (d) determining the affinity of the neoepitope tothe MHC molecule of the individual; optionally (e) determining theaffinity of the complex comprising the neoepitope and the MHC moleculeto a T cell receptor; (f) incorporating a neoantigen peptide comprisingthe neoepitope in a plurality of tumor antigen peptides; (g) contactinga population of PBMCs with the plurality of tumor antigen peptides toobtain a population of activated PBMCs (such as in the presence of animmune checkpoint inhibitor); and (h) administering to the individual aneffective amount of the activated PBMCs. In some embodiments, theactivated PBMCs are administered for at least three times. In someembodiments, the interval between each administration of the activatedPBMCs is about 2 weeks to about 5 months (such as about 3 months). Insome embodiments, the activated PBMCs are administered intravenously. Insome embodiments, the population of PBMCs is obtained from theindividual being treated. In some embodiments, the plurality of tumorantigen peptides comprises at least 10 tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises a firstcore group of general tumor antigen peptides and optionally a secondgroup of cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a plurality of neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the individual isselected for the method of treating based on having a low mutation load(such as in one or more MEW genes) in the cancer.

Monitoring after MASCT

Any of the MASCT methods described herein may further comprise amonitoring step after the individual receives the MASCT. Post-treatmentmonitoring may be beneficial for adjusting the treatment regimen of theindividual to optimize treatment outcome.

For example, the plurality of tumor antigen peptides described hereinmay be adjusted or customized based on the specific immune response ofthe individual against each of the plurality of tumor antigen peptidesand/or the clinical response of the individual to the activated T cellsor activated PBMCs in order to provide a plurality of customized tumorantigen peptides, which may be used for repeated MASCT treatment(s). Insome embodiments, tumor antigen peptides that do not elicit a strongspecific immune response can be removed from the antigen peptide poolfor future preparations of the pulsed DCs, activated T cells, oractivated PBMCs. In some embodiments, if the individual does not respond(such as having signs of disease progression, metastasis, etc.) to theMASCT treatment using one antigen peptide pool, the antigen peptide poolmay be adjusted, or neoantigens may be incorporated in the antigenpeptide pool for use in a second cycle of MASCT treatment.

Thus, in some embodiments, there is provided a method of treating acancer in an individual, comprising: (a) optionally administering to theindividual an effective amount of dendritic cells loaded with aplurality of tumor antigen peptides; (b) administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof dendritic cells loaded with the plurality of tumor antigen peptides;and (c) monitoring the individual after the administration of theactivated T cells. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the co-culturing is for about 7 days toabout 21 days (such as about 7 days to about 14 days, or about 14 daysto about 21 days). In some embodiments, the population of T cell iscontacted with an immune checkpoint inhibitor (such as an inhibitor ofPD-1, PD-L1, or CTLA-4) prior to and/or during the co-culturing. In someembodiments, the activated T cells are administered intravenously. Insome embodiments, the activated T cells are administered for at leastthree times. In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the population of dendritic cells and the population of Tcells are derived from the same individual, such as the individual beingtreated. In some embodiments, the plurality of tumor antigen peptidescomprises at least 10 tumor antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides and optionally a second group ofcancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the individual isselected for the method of treating based on having a low mutation load(such as in one or more MEW genes) in the cancer. In some embodiments,the individual is selected for the method of treating based on havingone or more (such as at least 5) neoantigens in the individual. In someembodiments, the method further comprises identifying a neoantigen ofthe individual (such as by sequencing a tumor sample from theindividual), and incorporating a neoantigen peptide in the plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen. In some embodiments, the method furthercomprises monitoring the individual after the administration of theactivated T cells. In some embodiments, the monitoring comprisesdetermining the number of circulating tumor cells (CTC) in theindividual. In some embodiments, the monitoring comprises detecting aspecific immune response against the plurality of tumor antigen peptidesin the individual. In some embodiments, the plurality of tumor antigenpeptides is adjusted based on the specific immune response to provide aplurality of customized tumor antigen peptides. In some embodiments, themethod is repeated using the plurality of customized tumor antigenpeptides.

In some embodiments, there is provided a method of monitoring atreatment in an individual having a cancer with activated T cells,comprising determining the number of circulating tumor cells (CTC) inthe individual, and/or detecting a specific immune response against eachof a plurality of tumor antigen peptides in the individual, wherein theactivated T cells are obtained by co-culturing a population of T cellswith a population of dendritic cells loaded with the plurality of tumorantigen peptides, and wherein the treatment comprises optionallyadministering to the individual an effective amount of the dendriticcells loaded with the plurality of tumor antigen peptides, andadministering to the individual an effective amount of the activated Tcells. In some embodiments, the interval between the administration ofthe dendritic cells and the administration of the activated T cells isabout 7 days to about 21 days (such as about 7 days to about 14 days,about 14 days to about 21 days, about 10 days or about 14 days). In someembodiments, the co-culturing is for about 7 days to about 21 days (suchas about 7 days to about 14 days, or about 14 days to about 21 days). Insome embodiments, the population of T cell is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4)prior to and/or during the co-culturing. In some embodiments, theactivated T cells are administered intravenously. In some embodiments,the activated T cells are administered for at least three times. In someembodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, thepopulation of dendritic cells and the population of T cells are derivedfrom the same individual, such as the individual being treated. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and optionally a second group of cancer-type specific antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises one or more neoantigen peptides. In some embodiments, themethod further comprises administering to the individual an effectiveamount of an immune checkpoint inhibitor, such as an inhibitor of PD-1,PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments,the individual is selected for the method of treating based on having alow mutation load (such as in one or more MEW genes) in the cancer. Insome embodiments, the method further comprises selecting the individualfor the method of treating based on having one or more (such as at least5) neoantigens in the individual. In some embodiments, the methodfurther comprises identifying a neoantigen of the individual (such as bysequencing a tumor sample from the individual), providing a neoantigenpeptide based on the neoantigen, and incorporating the neoantigenpeptide in the plurality of tumor antigen peptides. In some embodiments,the plurality of tumor antigen peptides is adjusted based on thespecific immune response to provide a plurality of customized tumorantigen peptides. In some embodiments, the treatment is repeated usingthe plurality of customized tumor antigen peptides.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) inducing differentiation of a populationof monocytes into a population of dendritic cells (such as in thepresence of GM-CSF and IL-4); (b) contacting the population of dendriticcells with a plurality of tumor antigen peptides (such as in thepresence of a plurality of Toll-like Receptor (TLR) agonists) to obtaina population of dendritic cells loaded with the plurality of tumorantigen peptides; (c) optionally administering to the individual aneffective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing (such as in the presence of aplurality of cytokines and optionally an anti-CD3 antibody) thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells; (e) administering to the individual an effectiveamount of the activated T cells; and (f) monitoring the individual afterthe administration of the activated T cells, wherein the population ofmonocytes and the population of non-adherent PBMCs are obtained from apopulation of PBMCs (such as from the individual). In some embodiments,the interval between the administration of the dendritic cells and theadministration of the activated T cells is about 7 days to about 21 days(such as about 7 days to about 14 days, about 14 days to about 21 days,about 10 days or about 14 days). In some embodiments, the co-culturingis for about 7 days to about 21 days (such as about 7 days to about 14days, or about 14 days to about 21 days). In some embodiments, thepopulation of non-adherent PBMCs is contacted with an immune checkpointinhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) prior toand/or during the co-culturing. In some embodiments, the activated Tcells are administered intravenously. In some embodiments, the activatedT cells are administered for at least three times. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered subcutaneously. In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered for at least three times. In some embodiments, thepopulation of PBMCs is obtained from the individual being treated. Insome embodiments, the plurality of tumor antigen peptides comprises atleast 10 tumor antigen peptides. In some embodiments, the plurality oftumor antigen peptides comprises a first core group of general tumorantigen peptides and optionally a second group of cancer-type specificantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises one or more neoantigen peptides. In some embodiments,the method further comprises administering to the individual aneffective amount of an immune checkpoint inhibitor, such as an inhibitorof PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In someembodiments, the individual is selected for the method of treating basedon having a low mutation load (such as in one or more MEW genes) in thecancer. In some embodiments, the individual is selected for the methodof treating based on having one or more (such as at least 5) neoantigensin the individual. In some embodiments, the method further comprisesidentifying a neoantigen of the individual (such as by sequencing atumor sample from the individual), and incorporating a neoantigenpeptide in the plurality of tumor antigen peptides, wherein theneoantigen peptide comprises a neoepitope in the neoantigen. In someembodiments, the method further comprises monitoring the individualafter the administration of the activated T cells. In some embodiments,the monitoring comprises determining the number of circulating tumorcells (CTC) in the individual. In some embodiments, the monitoringcomprises detecting a specific immune response against the plurality oftumor antigen peptides in the individual. In some embodiments, theplurality of tumor antigen peptides is adjusted based on the specificimmune response to provide a plurality of customized tumor antigenpeptides. In some embodiments, the method is repeated using theplurality of customized tumor antigen peptides.

In some embodiments, there is provided a method of monitoring atreatment in an individual having a cancer with activated T cells,comprising determining the number of circulating tumor cells (CTC) inthe individual, and/or detecting a specific immune response against eachof a plurality of tumor antigen peptides in the individual, wherein theactivated T cells are obtained by steps comprising: (a) inducingdifferentiation of a population of monocytes into a population ofdendritic cells (such as in the presence of GM-CSF and IL-4); (b)contacting the population of dendritic cells with a plurality of tumorantigen peptides (such as in the presence of a plurality of Toll-likeReceptor (TLR) agonists) to obtain a population of dendritic cellsloaded with the plurality of tumor antigen peptides; and (c)co-culturing (such as in the presence of a plurality of cytokines andoptionally an anti-CD3 antibody) the population of dendritic cellsloaded with the plurality of tumor antigen peptides and a population ofnon-adherent PBMCs to obtain the population of activated T cells,wherein the population of monocytes and the population of non-adherentPBMCs are obtained from a population of PBMCs (such as from theindividual); and wherein the treatment comprises optionallyadministering to the individual an effective amount of the dendriticcells loaded with the plurality of tumor antigen peptides, andadministering to the individual an effective amount of the activated Tcells. In some embodiments, the interval between the administration ofthe dendritic cells and the administration of the activated T cells isabout 7 days to about 21 days (such as about 7 days to about 14 days,about 14 days to about 21 days, about 10 days or about 14 days). In someembodiments, the co-culturing is for about 7 days to about 21 days (suchas about 7 days to about 14 days, or about 14 days to about 21 days). Insome embodiments, the population of non-adherent PBMCs is contacted withan immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, orCTLA-4) prior to and/or during the co-culturing. In some embodiments,the activated T cells are administered intravenously. In someembodiments, the activated T cells are administered for at least threetimes. In some embodiments, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times. In someembodiments, the population of PBMCs is obtained from the individualbeing treated. In some embodiments, the plurality of tumor antigenpeptides comprises at least 10 tumor antigen peptides. In someembodiments, the plurality of tumor antigen peptides comprises a firstcore group of general tumor antigen peptides and optionally a secondgroup of cancer-type specific antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises one or more neoantigenpeptides. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the individual isselected for the method of treating based on having a low mutation load(such as in one or more MHC genes) in the cancer. In some embodiments,the method further comprises selecting the individual for the method oftreating based on having one or more (such as at least 5) neoantigens inthe individual. In some embodiments, the method further comprisesidentifying a neoantigen of the individual (such as by sequencing atumor sample from the individual), providing a neoantigen peptide basedon the neoantigen, and incorporating the neoantigen peptide in theplurality of tumor antigen peptides. In some embodiments, the pluralityof tumor antigen peptides is adjusted based on the specific immuneresponse to provide a plurality of customized tumor antigen peptides. Insome embodiments, the treatment is repeated using the plurality ofcustomized tumor antigen peptides.

In some embodiments, there is provided a method of treating a cancer inan individual, comprising: (a) contacting a population of PBMCs with aplurality of tumor antigen peptides (such as in the presence of animmune checkpoint inhibitor) to obtain a population of activated PBMCs;(b) administering to the individual an effective amount of the activatedPBMCs; and (c) monitoring the individual after the administration of theactivated PBMCs. In some embodiments, the activated PBMCs areadministered for at least three times. In some embodiments, the intervalbetween each administration of the activated PBMCs is about 2 weeks toabout 5 months (such as about 3 months). In some embodiments, theactivated PBMCs are administered intravenously. In some embodiments, thepopulation of PBMCs is obtained from the individual being treated. Insome embodiments, the plurality of tumor antigen peptides comprises atleast 10 tumor antigen peptides. In some embodiments, the plurality oftumor antigen peptides comprises a first core group of general tumorantigen peptides and optionally a second group of cancer-type specificantigen peptides. In some embodiments, the plurality of tumor antigenpeptides comprises one or more neoantigen peptides. In some embodiments,the method further comprises administering to the individual aneffective amount of an immune checkpoint inhibitor, such as an inhibitorof PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In someembodiments, the individual is selected for the method of treating basedon having a low mutation load (such as in one or more MHC genes) in thecancer. In some embodiments, the individual is selected for the methodof treating based on having one or more (such as at least 5) neoantigensin the individual. In some embodiments, the method further comprisesidentifying a neoantigen of the individual (such as by sequencing atumor sample from the individual), and incorporating a neoantigenpeptide in the plurality of tumor antigen peptides, wherein theneoantigen peptide comprises a neoepitope in the neoantigen. In someembodiments, the method further comprises monitoring the individualafter the administration of the activated PBMCs. In some embodiments,the monitoring comprises determining the number of circulating tumorcells (CTC) in the individual. In some embodiments, the monitoringcomprises detecting a specific immune response against the plurality oftumor antigen peptides in the individual. In some embodiments, theplurality of tumor antigen peptides is adjusted based on the specificimmune response to provide a plurality of customized tumor antigenpeptides. In some embodiments, the method is repeated using theplurality of customized tumor antigen peptides.

In some embodiments, there is provided a method of monitoring atreatment in an individual having a cancer with activated PBMCs,comprising determining the number of circulating tumor cells (CTC) inthe individual, and/or detecting a specific immune response against eachof a plurality of tumor antigen peptides in the individual, wherein theactivated PBMCs are obtained by contacting a population of PBMCs with aplurality of tumor antigen peptides (such as in the presence of animmune checkpoint inhibitor), and wherein the treatment comprisesadministering to the individual an effective amount of the activatedPBMCs. In some embodiments, the activated PBMCs are administered for atleast three times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises oneor more neoantigen peptides. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MHC genes) in the cancer. In someembodiments, the method further comprises selecting the individual forthe method of treating based on having one or more (such as at least 5)neoantigens in the individual. In some embodiments, the method furthercomprises identifying a neoantigen of the individual (such as bysequencing a tumor sample from the individual), providing a neoantigenpeptide based on the neoantigen, and incorporating the neoantigenpeptide in the plurality of tumor antigen peptides. In some embodiments,the plurality of tumor antigen peptides is adjusted based on thespecific immune response to provide a plurality of customized tumorantigen peptides. In some embodiments, the treatment is repeated usingthe plurality of customized tumor antigen peptides.

Specific immune response against an individual tumor antigen peptide maybe determined using any known methods in the art, for example, bymeasuring levels of cytotoxic factor (such as perforin or granzyme B),or cytokine release (such as IFNγ or TNFα) from T cells (or PBMCs) afterstimulation by the individual tumor antigen peptide. An antibody-basedassay, such as ELISPOT, may be used to quantify the cytotoxic factor, orcytokine (such as IFNγ) levels. Exemplary embodiments of the ELISPOTassay are described in the Examples. In some embodiments, the cytokine(such as IFNγ) release level from T cells (or PBMCs) in response to atumor antigen peptide is normalized to a reference, such as a baselinecytokine release level, or a nonspecific cytokine release level of fromT cells (or PBMCs) in response to an irrelevant peptide, to provide acytokine (such as IFNγ) fold change value. In some embodiments, acytokine (such as IFNγ) fold change value of more than about any of 1.2,1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, or more in an ELISPOT assay indicatestrong specific immune response against the tumor antigen peptide. Insome embodiments, a tumor antigen peptide with a cytokine (such as IFNγ)fold change value of less than about any of 10, 8, 6, 5, 4, 3, 2.5, 2,1.5, 1.2 or less in an ELISPOT assay is removed from the plurality oftumor antigen peptides to provide a plurality of customized tumorantigen peptides for future MASCT.

Clinical response of the individual to MASCT methods may be assessed byknown methods in the art by a physician, such as by imaging methods,blood tests, biomarker assessment, and biopsy. In some embodiments, theclinical response is monitored by determining the number of circulatingtumor cells (CTC) in the individual before and after receiving MASCT. Insome embodiments, the CTCs have detached from a primary tumor andcirculate in a bodily fluid. In some embodiments, the CTCs have detachedfrom a primary tumor and circulate in the bloodstream. In someembodiments, the CTCs are an indication of metastasis. CTC numbers canbe determined by a variety of methods known in the art, including, butnot limited to, CellSearch method, Epic Science method, isoflux, andmaintrac. In some embodiments, the number of single CTCs, includingspecific subtypes of CTCs, in a blood sample of the individual isdetermined. In some embodiments, a number of more than about any of 10,20, 50, 100, 150, 200, 300 or more of single CTCs per mL of the bloodsample in the individual after receiving MASCT indicates an increasedrisk of metastasis, and/or poor clinical response to MASCT. In someembodiments, an increased number (such as at least about any of 1.5, 2,3, 4, 5, 10, or more fold increase) of single CTCs of the individualafter receiving MASCT compared to before receiving MASCT indicates poorclinical response to MASCT. In some embodiments, the number of CTCclusters in a blood sample of the individual is determined. In someembodiments, detection of at least about any of 1, 5, 10, 50, 100, ormore CTC clusters in a blood sample of the individual after receivingMASCT indicates an increased risk of metastasis, and/or poor clinicalresponse to MASCT. In some embodiments, an increased number (such as atleast about any of 1.5, 2, 3, 4, 5, 10, or more fold increase) of CTCclusters of the individual after receiving MASCT compared to beforereceiving MASCT indicates poor clinical response to MASCT.

Dosing and Method of Administration

Generally, dosages, schedules, and routes of administration of theactivated T cells, the population of dendritic cells loaded with theplurality of tumor antigen peptides, and the activated PBMCs may bedetermined according to the size and condition of the individual, andaccording to standard pharmaceutical practice. Exemplary routes ofadministration include intravenous, intra-arterial, intraperitoneal,intrapulmonary, intravesicular, intramuscular, intra-tracheal,subcutaneous, intraocular, intrathecal, or transdermal. In someembodiments of the MASCT method, the dendritic cells loaded with theplurality of tumor antigen peptides are administered subcutaneously. Insome embodiments of the MASCT method, the activated T cells areadministered intravenously. In some embodiments of the PBMC-based MASCTmethod, the activated PBMCs are administered intravenously.

The dose of the cells administered to an individual may vary accordingto, for example, the particular type of cells being administered, theroute of administration, and the particular type and stage of cancerbeing treated. The amount should be sufficient to produce a desirableresponse, such as a therapeutic response against cancer, but withoutsevere toxicity or adverse events. In some embodiments, the amount ofthe activated T cells or the dendritic cells to be administered is atherapeutically effective amount. In some embodiments, the amount of thecells (such as multiple-antigen loaded dendritic cells, the activated Tcells, or the activated PBMCs) is an amount sufficient to decrease thesize of a tumor, decrease the number of cancer cells, or decrease thegrowth rate of a tumor by at least about any of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100% compared to the corresponding tumorsize, number of cancer cells, or tumor growth rate in the sameindividual prior to treatment or compared to the corresponding activityin other individuals not receiving the treatment. Standard methods canbe used to measure the magnitude of this effect, such as in vitro assayswith purified enzyme, cell-based assays, animal models, or humantesting.

In some embodiments, the population of dendritic cells loaded with theplurality of tumor antigen peptides are administered at a dose at leastabout any of 1×10⁵, 5×10⁵, 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷ or 5×10⁷ cells/individual. In someembodiments, the population of dendritic cells loaded with the pluralityof tumor antigen peptides are administered at a dose about any of1×10⁵-5×10⁵, 5×10⁵-1×10⁶, 1×10⁶-2×10⁶, 2×10⁶-3×10⁶, 3×10⁶-4×10⁶,4×10⁶-5×10⁶, 5×10⁶-6×10⁶, 6×10⁶-7×10⁶, 7×10⁶-8×10⁶, 8×10⁶-1×10⁸,1×10⁶-3×10⁶, 3×10⁶-5×10⁶, 5×10⁶-7×10⁶, 2×10⁶-4×10⁶, 1×10⁶-5×10⁶, or5×10⁶-1×10⁷ cells/individual. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered ata dose of at least about 1×10⁶ cells/individual. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered at a dose of about 1×10⁶ to about 5×10⁶cells/individual.

In some embodiments, the population of dendritic cells loaded with theplurality of tumor antigen peptides are administered at a dose at leastabout any of 1×10⁴, 5×10⁴, 1×10⁵, 2×10⁵, 4×10⁵, 6×10⁵, 8×10⁵, 1×10⁶,2×10⁶ or 1×10⁷ cells/kg. In some embodiments, the population ofdendritic cells loaded with the plurality of tumor antigen peptides areadministered at a dose about any of 1×10⁴-5×10⁴, 5×10⁴-1×10⁵,1×10⁵-2×10⁵, 2×10⁵-4×10⁵, 4×10⁵-6×10⁵, 6×10⁵-8×10⁵, 8×10⁵-1×10⁶,1×10⁶-2×10⁶, 2×10⁶-1×10⁷, 1×10⁴-1×10⁵, 1×10⁵-1×10⁶, 1×10⁶-1×10⁷,1×10⁴-1×10⁶, or 1×10⁵-1×10⁷ cells/kg. In some embodiments, the dendriticcells loaded with the plurality of tumor antigen peptides areadministered at a dose of at least about 2×10⁵ cells/kg. In someembodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered at a dose of about 2×10⁵ to about1×10⁶ cells/kg.

In some embodiments, the activated T cells are administered at a dose ofat least about any of 1×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 1.5×10¹⁰, 2×10¹⁰, or 5×10¹⁰cells/individual. In some embodiments, the activated T cells areadministered at a dose of any of about 1×10⁸ to about 5×10⁸, about 5×10⁸to about 9×10⁸, about 9×10⁸ to about 1×10⁹, about 1×10⁹ to about 2×10⁹,about 2×10⁹ to about 3×10⁹, about 3×10⁹ to about 4×10⁹, about 4×10⁹ toabout 5×10⁹, about 5×10⁹ to about 6×10⁹, about 6×10⁹ to about 1×10¹⁰,about 1×10⁹ to about 3×10⁹, about 3×10⁹ to about 5×10⁹, about 5×10⁹ toabout 7×10⁹, about 7×10⁹ to about 1×10¹⁰, about 1×10⁹ to about 5×10⁹,about 5×10⁹ to about 1×10¹⁰, about 3×10⁹ to about 7×10⁹, about 1×10¹⁰ toabout 1.5×10¹⁰, about 1×10¹⁰ to about 2×10¹⁰, or about 1×10⁹ to about1×10¹⁰ cells/individual. In some embodiments, the activated T cells areadministered at a dose of at least about 3×10⁹ cells/individual. In someembodiments, the activated T cells are administered at a dose of about1×10⁹ to about 1×10¹⁰ cells/individual.

In some embodiments, the activated T cells are administered at a dose ofat least about any of 1×10⁷, 1×10⁸, 2×10⁸, 4×10⁸, 6×10⁸, 8×10⁸, 1×10⁹,2×10⁹, 4×10⁹, 6×10⁹, 8×10⁹, 1×10¹⁰ cells/kg. In some embodiments, theactivated T cells are administered at a dose of any of about 1×10⁷ toabout 1×10⁸, about 1×10⁸ to about 2×10⁸, about 2×10⁸ to about 4×10⁸,about 4×10⁸ to about 6×10⁸, about 6×10⁸ to about 8×10⁸, about 8×10⁸ toabout 1×10⁹, about 1×10⁹ to about 2×10⁹, about 2×10⁹ to about 4×10⁹,about 4×10⁹ to about 1×10¹⁰, about 2×10⁸ to about 6×10⁸, about 6×10⁸ toabout 1×10⁹, about 1×10⁸ to about 2×10⁸, about 2×10⁸ to about 2×10⁹,about 1×10⁷ to about 1×10⁸, about 1×10⁸ to about 1×10⁹, about 1×10⁹ toabout 1×10¹⁰, or about 1×10⁷ to about 1×10⁹ cells/kg. In someembodiments, the activated T cells are administered at a dose of atleast about 6×10⁸ cells/kg. In some embodiments, the activated T cellsare administered at a dose of about 2×10⁸ to about 2×10⁹ cells/kg.

In some embodiments, the activated PBMCs are administered at a dose ofat least about any of 1×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹,6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 1.5×10¹⁰, 2×10¹⁰, or 5×10¹⁰cells/individual. In some embodiments, the activated PBMCs areadministered at a dose of any of about 1×10⁸ to about 5×10⁸, about 5×10⁸to about 9×10⁸, about 9×10⁸ to about 1×10⁹, about 1×10⁹ to about 2×10⁹,about 2×10⁹ to about 3×10⁹, about 3×10⁹ to about 4×10⁹, about 4×10⁹ toabout 5×10⁹, about 5×10⁹ to about 6×10⁹, about 6×10⁹ to about 1×10¹⁰,about 1×10⁹ to about 3×10⁹, about 3×10⁹ to about 5×10⁹, about 5×10⁹ toabout 7×10⁹, about 7×10⁹ to about 1×10¹⁰, about 1×10⁹ to about 5×10⁹,about 5×10⁹ to about 1×10¹⁰, about 3×10⁹ to about 7×10⁹, about 1×10¹⁰ toabout 1.5×10¹⁰, about 1×10¹⁰ to about 2×10¹⁰, or about 1×10⁹ to about1×10¹⁰ cells/individual. In some embodiments, the activated PBMCs areadministered at a dose of at least about 1×10⁹ cells/individual. In someembodiments, the activated PBMCs are administered at a dose of about1×10⁹ to about 1×10¹⁰ cells/individual.

In some embodiments, the activated PBMCs are administered at a dose ofat least about any of 1×10⁷, 1×10⁸, 2×10⁸, 4×10⁸, 6×10⁸, 8×10⁸, 1×10⁹,2×10⁹, 4×10⁹, 6×10⁹, 8×10⁹, 1×10¹⁰ cells/kg. In some embodiments, theactivated PBMCs are administered at a dose of any of about 1×10⁷ toabout 1×10⁸, about 1×10⁸ to about 2×10⁸, about 2×10⁸ to about 4×10⁸,about 4×10⁸ to about 6×10⁸, about 6×10⁸ to about 8×10⁸, about 8×10⁸ toabout 1×10⁹, about 1×10⁹ to about 2×10⁹, about 2×10⁹ to about 4×10⁹,about 4×10⁹ to about 1×10¹⁰, about 2×10⁸ to about 6×10⁸, about 6×10⁸ toabout 1×10⁹, about 1×10⁸ to about 2×10⁸, about 2×10⁸ to about 2×10⁹,about 1×10⁷ to about 1×10⁸, about 1×10⁸ to about 1×10⁹, about 1×10⁹ toabout 1×10¹⁰, or about 1×10⁷ to about 1×10⁹ cells/kg. In someembodiments, the activated PBMCs are administered at a dose of about2×10⁸ to about 2×10⁹ cells/kg.

In some embodiments, a stabilizing agent or an excipient, such as humanalbumin, is used together with the activated T cells, the population ofdendritic cells loaded with the plurality of tumor antigen peptides,and/or the activated PBMC cells when administered.

The dosage and dosing schedule of the cells in the MASCT method(including the PBMC-based MASCT method) may be adjusted over the courseof the treatment, based on the judgment of the administering physician.In some embodiments, the activated T cells are administered about anyone of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17days, 18 days, 19 days, 20 days, 21 days, or 1 month, after thedendritic cells loaded with the plurality of tumor antigen peptides areadministered. In some embodiments, the activated T cells areadministered concurrently with the dendritic cells. In some embodiments,the activated T cells are administered about 14-21 days after thedendritic cells are administered. In some exemplary embodiments, theactivated T cells are administered about 14 days after the dendriticcells are administered. Exemplary embodiments of the MASCT methods withexemplary schedule of administration are shown in FIG. 1 and FIG. 2A.

The MASCT method (including the PBMC-based MASCT method, and precisionMASCT method) may involve a single treatment, or repeated treatments. Insome embodiments, the activated T cells are administered for any one of1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In someembodiments, the activated T cells are administered at least 3 times. Insome embodiments, the dendritic cells are administered for any one of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. In some embodiments,the dendritic cells are administered at least 3 times. In someembodiments of the PBMC-based MASCT method, the activated PBMCs areadministered for any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than10 times. In some embodiments of the PBMC-based MASCT method, theactivated PBMCs are administered at least 3 times. In some embodiments,one or more cell (such as antigen-loaded dendritic cell or activated Tcells) preparation steps are repeated prior to the repeatedadministration of the dendritic cells, the activated T cells, or both.In some embodiments, the MASCT method (including the PBMC-based MASCTmethod, and precision MASCT method) is repeated once per week, once 2weeks, once 3 weeks, once 4 weeks, once per month, once per 2 months,once per 3 months, once per 4 months, once per 5 months, once per 6months, once per 7 months, once per 8 months, once per 9 months, or onceper year. In some embodiments, the interval between each administrationof the dendritic cells, the activated T cells, or the PBMCs is about anyone of 1 week to 2 weeks, 2 weeks to 1 month, 2 weeks to 2 months, 1month to 2 months, 1 month to 3 months, 3 months to 6 months, or 6months to a year. In some embodiments, the interval between eachadministration of the dendritic cells, the activated T cells, or thePBMCs is about 0.5 to about 5 months, such as about 2 weeks to about 2months, or about 2 months to about 5 months. In some exemplaryembodiments, all step(s) of the MASCT method (including the PBMC-basedMASCT method, and precision MASCT method) are repeated once per monthduring the first 6 months of treatment, every two months for the second6 months of treatment, and every half a year after first 12 months oftreatment if the individual has stable disease. Any embodiment of theMASCT method described herein (including the PBMC-based MASCT method,and precision MASCT method) can be combined with any other embodiment ofthe MASCT method (including the PBMC-based MASCT method, and precisionMASCT method) during the full course of a repeated treatment.

The MASCT method (including the PBMC-based MASCT method, and precisionMASCT method) provided herein may be used as a first therapy, secondtherapy, third therapy, or combination therapy with other types ofcancer therapies known in the art, such as chemotherapy, surgery,radiation, gene therapy, immunotherapy, bone marrow transplantation,stem cell transplantation, targeted therapy, cryotherapy, ultrasoundtherapy, photodynamic therapy, radio-frequency ablation or the like, inan adjuvant setting or a neoadjuvant setting. In some embodiments, thepresent invention provides a method of treating a cancer in anindividual comprising a first therapy comprising administering to theindividual an effective amount of activated T cells, wherein the T cellsare activated by co-culturing with a population of dendritic cellsloaded with a plurality of tumor antigen peptides. In some embodimentsof the method used as a first therapy, there exists no other approvedanti-cancer therapy for the individual. In some embodiments, the MASCTmethod (including the PBMC-based MASCT method, and precision MASCTmethod) is used as a second therapy, wherein the individual haspreviously received resection, radio-frequency ablation, chemotherapy,radiation therapy, or other types of cancer therapy. In someembodiments, the individual has progressed or has not been able totolerate standard anti-cancer therapy. In some embodiments, theindividual receives other types of cancer therapy prior to, concurrentlywith, or after the MASCT treatment(s). For example, the MASCT method(including the PBMC-based MASCT method, and precision MASCT method) mayprecede or follow the other cancer therapy (such as chemotherapy,radiation, surgery or combination thereof) by intervals ranging fromminutes, days, weeks to months. In some embodiments, the intervalbetween the first and the second therapy is such that the activated Tcells of the MASCT method (including the PBMC-based MASCT method, andprecision MASCT method) and the other cancer therapy (such aschemotherapy, radiation, surgery, or combination thereof) would be ableto exert an advantageously combined effect on the individual. In someembodiments, the MASCT method (including the PBMC-based MASCT method,and precision MASCT method) is used in conjunction with other cancertherapy (such as chemotherapy, radiation, surgery, or combinationthereof) treat cancer in an individual. The combination therapy methodsdescribed herein may be performed alone or in conjunction with anothertherapy, such as surgery, radiation, gene therapy, immunotherapy, bonemarrow transplantation, stem cell transplantation, hormone therapy,targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy,chemotherapy or the like. Additionally, a person having a greater riskof developing a proliferative disease may receive treatments to inhibitand/or delay the development of the disease.

In some embodiments, the method comprises a method of inhibiting cancercell proliferation (such as tumor growth) in an individual, comprisingadministering to the individual an effective amount of activated Tcells, wherein the activated T cells are prepared by co-culturing apopulation of T cells with a population of dendritic cells loaded with aplurality of tumor antigen peptides. In some embodiments, the methodcomprises a method of inhibiting cancer cell proliferation (such astumor growth) in an individual, comprising administering to theindividual an effective amount of dendritic cells loaded with aplurality of tumor antigen peptide, and administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof dendritic cells loaded with a plurality of tumor antigen peptides. Insome embodiments, the method comprises a method of inhibiting cancercell proliferation (such as tumor growth) in an individual, comprisingcontacting a population of PBMCs with a plurality of tumor antigenpeptides to obtain a population of activated PBMCs, and administering tothe individual an effective amount of the activated PBMCs. In someembodiments, the method further comprises administering to theindividual an effective amount of an immune checkpoint inhibitor, suchas an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, orLAG-3. In some embodiments, the activated T cells or PBMCs areadministered for at least three times. In some embodiments, at leastabout 10% (including for example at least about any of 20%, 30%, 40%,60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited.

In some embodiments, the method comprises a method of inhibiting tumormetastasis in an individual, comprising administering to the individualan effective amount of activated T cells, wherein the activated T cellsare prepared by co-culturing a population of T cells with a populationof dendritic cells loaded with a plurality of tumor antigen peptides. Insome embodiments, the method comprises a method of inhibiting tumormetastasis in an individual, comprising administering to the individualan effective amount of dendritic cells loaded with a plurality of tumorantigen peptide, and administering to the individual an effective amountof activated T cells, wherein the activated T cells are prepared byco-culturing a population of T cells with a population of dendriticcells loaded with a plurality of tumor antigen peptides. In someembodiments, the method comprises a method of inhibiting tumormetastasis in an individual, comprising contacting a population of PBMCswith a plurality of tumor antigen peptides to obtain a population ofactivated PBMCs, and administering to the individual an effective amountof the activated PBMCs. In some embodiments, the activated T cells orPBMCs are administered for at least three times. In some embodiments,the method further comprises administering to the individual aneffective amount of an immune checkpoint inhibitor, such as an inhibitorof PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In someembodiments, at least about 10% (including for example at least aboutany of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) metastasis isinhibited. In some embodiments, method of inhibiting metastasis to lymphnode is provided.

In some embodiments, the method comprises a method of reducing tumorsize in an individual, comprising administering to the individual aneffective amount of activated T cells, wherein the activated T cells areprepared by co-culturing a population of T cells with a population ofdendritic cells loaded with a plurality of tumor antigen peptides. Insome embodiments, the method comprises a method of reducing tumor sizein an individual, comprising administering to the individual aneffective amount of dendritic cells loaded with a plurality of tumorantigen peptide, and administering to the individual an effective amountof activated T cells, wherein the activated T cells are prepared byco-culturing a population of T cells with a population of dendriticcells loaded with a plurality of tumor antigen peptides. In someembodiments, the method comprises a method of reducing tumor size in anindividual, comprising contacting a population of PBMCs with a pluralityof tumor antigen peptides to obtain a population of activated PBMCs, andadministering to the individual an effective amount of the activatedPBMCs. In some embodiments, the activated T cells or PBMCs areadministered for at least three times. In some embodiments, the methodfurther comprises administering to the individual an effective amount ofan immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, thetumor size is reduced at least about 10% (including for example at leastabout any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%).

In some embodiments, the method comprises a method of prolongingprogression-free survival of cancer in an individual, comprisingadministering to the individual an effective amount of activated Tcells, wherein the activated T cells are prepared by co-culturing apopulation of T cells with a population of dendritic cells loaded with aplurality of tumor antigen peptides. In some embodiments, the methodcomprises a method of prolonging progression-free survival of cancer inan individual, comprising administering to the individual an effectiveamount of dendritic cells loaded with a plurality of tumor antigenpeptide, and administering to the individual an effective amount ofactivated T cells, wherein the activated T cells are prepared byco-culturing a population of T cells with a population of dendriticcells loaded with a plurality of tumor antigen peptides. In someembodiments, the method comprises a method of prolongingprogression-free survival of cancer in an individual, comprisingcontacting a population of PBMCs with a plurality of tumor antigenpeptides to obtain a population of activated PBMCs, and administering tothe individual an effective amount of the activated PBMCs. In someembodiments, the activated T cells or PBMCs are administered for atleast three times. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the method prolongsthe time to disease progression by at least any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 weeks.

In some embodiments, the method comprises a method of prolongingsurvival of an individual having cancer, comprising administering to theindividual an effective amount of activated T cells, wherein theactivated T cells are prepared by co-culturing a population of T cellswith a population of dendritic cells loaded with a plurality of tumorantigen peptides. In some embodiments, the method comprises a method ofprolonging survival of an individual having cancer, comprisingadministering to the individual an effective amount of dendritic cellsloaded with a plurality of tumor antigen peptide, and administering tothe individual an effective amount of activated T cells, wherein theactivated T cells are prepared by co-culturing a population of T cellswith a population of dendritic cells loaded with a plurality of tumorantigen peptides. In some embodiments, the method comprises a method ofprolonging survival of an individual having cancer, comprisingcontacting a population of PBMCs with a plurality of tumor antigenpeptides to obtain a population of activated PBMCs, and administering tothe individual an effective amount of the activated PBMCs. In someembodiments, the activated T cells or PBMCs are administered for atleast three times. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the method prolongsthe time to disease progression by at least any of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, or 12 weeks. In some embodiments, the method prolongs thesurvival of the individual by at least any of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 18, or 24 months.

In some embodiments of any of the methods, the method comprises a methodof reducing AEs and SAEs in an individual having cancer, comprisingadministering to the individual an effective amount of activated Tcells, wherein the activated T cells are prepared by co-culturing apopulation of T cells with a population of dendritic cells loaded with aplurality of tumor antigen peptides. In some embodiments of any of themethods, the method comprises a method of reducing AEs and SAEs in anindividual having cancer, comprising administering to the individual aneffective amount of dendritic cells loaded with a plurality of tumorantigen peptide, and administering to the individual an effective amountof activated T cells, wherein the activated T cells are prepared byco-culturing a population of T cells with a population of dendriticcells loaded with a plurality of tumor antigen peptides. In someembodiments of any of the methods, the method comprises a method ofreducing AEs and SAEs in an individual having cancer, comprisingcontacting a population of PBMCs with a plurality of tumor antigenpeptides to obtain a population of activated PBMCs, and administering tothe individual an effective amount of the activated PBMCs. In someembodiments, the activated T cells or PBMCs are administered for atleast three times. In some embodiments, the method further comprisesadministering to the individual an effective amount of an immunecheckpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO,TIM-3, BTLA, VISTA, or LAG-3.

In some embodiments, the method is predictive of and/or results in anobjective response (such as a partial response or complete response)._Insome embodiments, the method is predictive of and/or results in improvedquality of life.

Some cancer immunotherapies are associated with immune-related adverseevents (irAEs) in additional to common adverse events generallyassociated with other cancer therapies. IrAEs are usuallymechanistically related to either on-target T-cell toxicity againsttarget antigens that are expressed in normal, non-tumor tissue, socalled on-target off-tumor effect, or off-target effects such asbreaking of self-tolerance or epitope cross-reaction. IrAEs can lead tosevere symptoms and conditions on the dermatologic, gastrointestinal,endocrine, hepatic, ocular, neurologic, and other tissues or organs.Typical irAEs reported for cancer immunotherapy methods known in the artinclude fatal immune-mediated dermatitis, pneumonia, colitis,lymphocytic hypophysitis, pancreatitis, lymphadenopathy, endocrinedisorders, CNS toxicity, and the like. In some embodiments, the MASCTmethods (including the PBMC-based MASCT methods) described herein areassociated with low incidence of adverse events, such as irAEs. In someembodiments, less than about any one of 50%, 40%, 30%, 20%, 10%, 5%, 4%,3%, 2%, or 1% of individuals experience irAEs, such as irAEs of Grade2-5.

Immune Checkpoint Inhibitors

The MASCT methods in some embodiments use immune checkpoint inhibitors,for example, in the preparation of the activated T cells or PBMCs (suchas prior to and/or during the co-culturing step), and/or in combinationtherapy. Any known immune checkpoint inhibitors may be used, including,but not limited to the immune checkpoint inhibitors described in thissection.

In some embodiments, the immune checkpoint inhibitor is a natural orengineered ligand of an inhibitory immune checkpoint molecule,including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands ofTIM-3 (e.g., Galectin-9), ligands of A2a Receptor (e.g., adenosine,Regadenoson), ligands of LAG-3 (e.g., MHC class I or MHC class IImolecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g.,MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1,PD-L2), ligands of IDO (e.g., NKTR-218, Indoximod, NLG919), and ligandsof CD47 (e.g., SIRP-alpha receptor).

The immune checkpoint inhibitors may be of any suitable molecularmodality, including, but not limited to, small molecules, nucleic acids(such as DNA, RNAi, or aptamer), peptides, or proteins (such asantibodies).

In some embodiments, the immune checkpoint inhibitor is an antibody(such as antagonist antibody) that targets an inhibitory immunecheckpoint protein selected from the group consisting of anti-CTLA-4(e.g., Ipilimumab, Tremelimumab, KAHR-102), anti-TIM-3 (e.g., F38-2E2,ENUM005), anti-LAG-3 (e.g., BMS-986016, IMP701, IMP321, C9B7W), anti-KIR(e.g., Lirilumab and IPH2101), anti-PD-1 (e.g., Nivolumab, Pidilizumab,Pembrolizumab, BMS-936559, atezolizumab, Pembrolizumab, MK-3475,AMP-224, AMP-514, STI-A1110, TSR-042), anti-PD-L1 (e.g., KY-1003(EP20120194977), MCLA-145, RG7446, BMS-936559, MEDI-4736, MSB0010718C,AUR-012, STI-A1010, PCT/US2001/020964, MPDL3280A, AMP-224, Dapirolizumabpegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42 (OSU-HDAC42,HDAC-42, AR42, AR 42, OSU-HDAC 42, OSU-HDAC-42, NSC D736012, HDAC-42,HDAC 42, HDAC42, NSCD736012, NSC-D736012), MEDI-9447), anti-B7-H3 (e.g.,MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002, TTI-621, VLST-007),anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (suchas alemtuzumab), anti-IL-10, anti-IL-35, and anti-TGF-β (such asFresolumimab). In some embodiments, the antibody is a monoclonalantibody. In some embodiments, the antibody is a full-length antibody.In some embodiments, the antibody is an antigen-binding fragmentselected from the group consisting of Fab, Fab′, F(ab′)₂, Fv, scFv,BiTE, nanobody, and other antigen-binding subsequences of the fulllength antibody or engineered combinations thereof. In some embodiments,the antibody is a human antibody, a humanized antibody, or a chimericantibody. In some embodiments, the antibody is a bispecific ormultispecific antibody.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-1. In some embodiments, the immune checkpoint inhibitor is ananti-PD-1 antibody. Exemplary anti-PD-1 antibodies include, but are notlimited to, Nivolumab, pembrolizumab, pidilizumab, BMS-936559, andatezolizumab, Pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, andTSR-042. In some embodiments, the immune checkpoint inhibitor isnivolumab (for example, OPDIVO®). In some embodiments, the immunecheckpoint inhibitor is Pembrolizumab (for example, KEYTRUDA®). In someembodiments, the immune checkpoint inhibitor is SHR-1210.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofPD-L1. In some embodiments, the immune checkpoint inhibitor is ananti-PD-L1 antibody. Exemplary anti-PD-L1 antibodies include, but arenot limited to, KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A,MEDI4736, Avelumab, or STI-A1010.

In some embodiments, the immune checkpoint inhibitor is an inhibitor ofCTLA-4. In some embodiments, the immune checkpoint inhibitor is ananti-CTLA-4 antibody. Exemplary anti-CTLA-4 antibodies include, but arenot limited to, Ipilimumab, Tremelimumab, and KAHR-102. In someembodiments, the immune checkpoint inhibitor is Ipilimumab (for example,YERVOY®).

A suitable concentration of the immune checkpoint inhibitor in theculturing media include, but are not limited to, at least about any of 1μg/mL, 10 μg/mL, 15 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 200 μg/mL, 500μg/mL, or 1 mg/mL. In some embodiments, the concentration of the immunecheckpoint inhibitor in the culturing media is any one of about 1 μg/mLto about 10 μg/mL, about 10 μg/mL to about 25 μg/mL, about 25 μg/mL toabout 50 μg/mL, about 50 μg/mL to about 100 μg/mL, about 100 μg/mL toabout 200 μg/mL, about 200 μg/mL to about 500 μg/mL, about 100 μg/mL toabout 1 mg/mL, about 10 μg/mL to about 100 μg/mL, or about 1 μg/mL toabout 100 μg/mL.

Any of the above MASCT methods (including PMBC-based MASCT methods andprecision MASCT methods) can be applied in combination withadministration of one or more immune checkpoint inhibitors. Exemplaryroutes of administration of the immune checkpoint inhibitor include, butare not limited to, intratumoral, intravesical, intramuscular,intraperitoneal, intravenous, intra-arterial, intracranial,intrapleural, subcutaneous, and epidermal routes, or be delivered intolymph glands, body spaces, organs or tissues known to contain such livecancer cells. In some embodiments, the immune checkpoint inhibitor isadministered intravenously. In some embodiments, the immune checkpointinhibitor is administered by infusion. In some embodiments, the immunecheckpoint inhibitor is infused over at least about any of 10 minutes,30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, or more. In someembodiments, the immune checkpoint inhibitor is administered via thesame administration route as the activated T cells or the activatedPBMCs. In some embodiments, the immune checkpoint inhibitor isadministered via a different administration route as the activated Tcells or the activated PBMCs.

Suitable dose of the immune checkpoint inhibitor include, but are notlimited to, about any one of 1 mg/m², 5 mg/m², 10 mg/m², 20 mg/m², 50mg/m², 100 mg/m², 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 750 mg/m²,1000 mg/m², or more. In some embodiments, the dose of immune checkpointinhibitor is any one of about 1 to about 5 mg/m², about 5 to about 10mg/m², about 10 to about 20 mg/m², about 20 to about 50 mg/m², about 50to about 100 mg/m², about 100 mg/m² to about 200 mg/m², about 200 toabout 300 mg/m², about 300 to about 400 mg/m², about 400 to about 500mg/m², about 500 to about 750 mg/m², or about 750 to about 1000 mg/m².In some embodiments, the dose of immune checkpoint inhibitor is aboutany one of 1 μg/kg, 2 μg/kg, 5 μg/kg, 10 μg/kg, 20 μg/kg, 50 μg/kg, 0.1mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 5mg/kg, 10 mg/kg, 20 mg/kg, 50 mg/kg, 100 mg/kg, or more. In someembodiments, the dose of the immune checkpoint inhibitor is any one ofabout 1 μg/kg to about 5 μg/kg, about 5 μg/kg to about 10 μg/kg, about10 μg/kg to about 50 μg/kg, about 50 μg/kg to about 0.1 mg/kg, about 0.1mg/kg to about 0.2 mg/kg, about 0.2 mg/kg to about 0.3 mg/kg, about 0.3mg/kg to about 0.4 mg/kg, about 0.4 mg/kg to about 0.5 mg/kg, about 0.5mg/kg to about 1 mg/kg, about 1 mg/kg to about 5 mg/kg, about 5 mg/kg toabout 10 mg/kg, about 10 mg/kg to about 20 mg/kg, about 20 mg/kg toabout 50 mg/kg, about 50 mg/kg to about 100 mg/kg, or about 1 mg/kg toabout 100 mg/kg.

In some embodiments, the immune checkpoint inhibitor is administereddaily. In some embodiments, the immune checkpoint inhibitor isadministered is administered at least about any one of 1×, 2×, 3×, 4×,5×, 6×, or 7× (i.e., daily) a week. In some embodiments, the immunecheckpoint inhibitor is administered weekly. In some embodiments, theimmune checkpoint inhibitor is administered weekly without break;weekly, two out of three weeks; weekly three out of four weeks; onceevery two weeks; once every 3 weeks; once every 4 weeks; once every 6weeks; once every 8 weeks, monthly, or every two to 12 months. In someembodiments, the intervals between each administration are less thanabout any one of 6 months, 3 months, 1 month, 20 days, 15, days, 12days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2days, or 1 day. In some embodiments, the intervals between eachadministration are more than about any one of 1 month, 2 months, 3months, 4 months, 5 months, 6 months, 8 months, or 12 months. In someembodiments, the immune checkpoint inhibitor is administered once every3 months. In some embodiments, there is no break in the dosing schedule.In some embodiments, the interval between each administration is no morethan about a week. In some embodiments, the immune checkpoint inhibitoris administered with the same dosing schedule as the activated T cellsor the activated PBMCs. In some embodiments, the immune checkpointinhibitor is administered with a different dosing schedule as theactivated T cells or the activated PBMCs.

In some embodiments, the immune checkpoint inhibitor is administered inevery MASCT treatment cycle. For example, the immune checkpointinhibitor may be administered about any of 1, 2, 3, 4, 5, 6, or moretimes every MASCT treatment cycle. In some embodiments, the immunecheckpoint inhibitor is not administered in every MASCT treatment cycle.For example, the immune checkpoint inhibitor may be administered aboutonce every 1, 2, 3, 4, 5, or more MASCT treatment cycles.

The administration of the immune checkpoint inhibitor can be over anextended period of time, such as from about a month up to about sevenyears. In some embodiments, the immune checkpoint inhibitor isadministered over a period of at least about any one of 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. Insome embodiments, the immune checkpoint inhibitor is administered for asingle time. In some embodiments, the immune checkpoint inhibitor isadministered repeatedly. In some embodiments, the immune checkpointinhibitor is administered repeatedly until disease progression.

T Cell Receptors (TCR)

The present invention in one aspect further provides a method of cloninga tumor-specific T cell receptor from an individual treated with any ofthe MASCT methods (including PBMC-based MASCT and precision MASCT)described herein.

Thus, in some embodiments, there is provided a method of cloning atumor-specific T cell receptor, comprising: (a) optionally administeringto an individual having a cancer an effective amount of dendritic cellsloaded with a plurality of tumor antigen peptides, (b) administering tothe individual an effective amount of activated T cells, wherein theactivated T cells are prepared by co-culturing a population of T cellswith a population of dendritic cells loaded with the plurality of tumorantigen peptides; (c) isolating a T cell from the individual, whereinthe T cell specifically recognizes a tumor antigen peptide in theplurality of tumor antigen peptides; and (d) cloning a T cell receptorfrom the T cell to provide the tumor-specific T cell receptor. In someembodiments, the interval between the administration of the dendriticcells and the administration of the activated T cells is about 7 days toabout 21 days (such as about 7 days to about 14 days, about 14 days toabout 21 days, about 10 days or about 14 days). In some embodiments, theco-culturing is for about 7 days to about 21 days (such as about 7 daysto about 14 days, or about 14 days to about 21 days). In someembodiments, the population of T cell is contacted with an immunecheckpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4)prior to and/or during the co-culturing. In some embodiments, theactivated T cells are administered intravenously. In some embodiments,the activated T cells are administered for at least three times. In someembodiments, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously. In some embodiments,the dendritic cells loaded with the plurality of tumor antigen peptidesare administered for at least three times. In some embodiments, thepopulation of dendritic cells and the population of T cells are derivedfrom the same individual, such as the individual being treated. In someembodiments, the plurality of tumor antigen peptides comprises at least10 tumor antigen peptides. In some embodiments, the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides and optionally a second group of cancer-type specific antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises one or more neoantigen peptides. In some embodiments, themethod further comprises administering to the individual an effectiveamount of an immune checkpoint inhibitor, such as an inhibitor of PD-1,PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments,the individual is selected for the method of treating based on having alow mutation load (such as in one or more MEW genes) in the cancer. Insome embodiments, the individual is selected for the method of treatingbased on having one or more (such as at least 5) neoantigens in theindividual. In some embodiments, the method further comprisesidentifying a neoantigen of the individual (such as by sequencing atumor sample from the individual), and incorporating a neoantigenpeptide in the plurality of tumor antigen peptides, wherein theneoantigen peptide comprises a neoepitope in the neoantigen. In someembodiments, the method further comprises monitoring the individualafter the administration of the activated T cells. In some embodiments,the monitoring comprises determining the number of circulating tumorcells (CTC) in the individual. In some embodiments, the monitoringcomprises detecting a specific immune response against the plurality oftumor antigen peptides in the individual. In some embodiments, theplurality of tumor antigen peptides is adjusted based on the specificimmune response to provide a plurality of customized tumor antigenpeptides. In some embodiments, the method is repeated using theplurality of customized tumor antigen peptides.

In some embodiments, there is provided a method of cloning atumor-specific T cell receptor, comprising: (a) inducing differentiationof a population of monocytes into a population of dendritic cells (suchas in the presence of GM-CSF and IL-4); (b) contacting the population ofdendritic cells with a plurality of tumor antigen peptides (such as inthe presence of a plurality of Toll-like Receptor (TLR) agonists) toobtain a population of dendritic cells loaded with the plurality oftumor antigen peptides; (c) optionally administering to the individualan effective amount of the dendritic cells loaded with the plurality oftumor antigen peptides; (d) co-culturing (such as in the presence of aplurality of cytokines and optionally an anti-CD3 antibody) thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides and a population of non-adherent PBMCs to obtain the populationof activated T cells; wherein the population of monocytes and thepopulation of non-adherent PBMCs are obtained from a population of PBMCs(such as from the individual); (e) administering to the individual aneffective amount of the activated T cells; (f) isolating a T cell fromthe individual, wherein the T cell specifically recognizes a tumorantigen peptide in the plurality of tumor antigen peptides; and (g)cloning a T cell receptor from the T cell to provide the tumor-specificT cell receptor. In some embodiments, the interval between theadministration of the dendritic cells and the administration of theactivated T cells is about 7 days to about 21 days (such as about 7 daysto about 14 days, about 14 days to about 21 days, about 10 days or about14 days). In some embodiments, the co-culturing is for about 7 days toabout 21 days (such as about 7 days to about 14 days, or about 14 daysto about 21 days). In some embodiments, the population of non-adherentPBMCs is contacted with an immune checkpoint inhibitor (such as aninhibitor of PD-1, PD-L1, or CTLA-4) prior to and/or during theco-culturing. In some embodiments, the activated T cells areadministered intravenously. In some embodiments, the activated T cellsare administered for at least three times. In some embodiments, thedendritic cells loaded with the plurality of tumor antigen peptides areadministered subcutaneously. In some embodiments, the dendritic cellsloaded with the plurality of tumor antigen peptides are administered forat least three times. In some embodiments, the population of PBMCs isobtained from the individual being treated. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises oneor more neoantigen peptides. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MEW genes) in the cancer. In someembodiments, the individual is selected for the method of treating basedon having one or more (such as at least 5) neoantigens in theindividual. In some embodiments, the method further comprisesidentifying a neoantigen of the individual (such as by sequencing atumor sample from the individual), and incorporating a neoantigenpeptide in the plurality of tumor antigen peptides, wherein theneoantigen peptide comprises a neoepitope in the neoantigen. In someembodiments, the method further comprises monitoring the individualafter the administration of the activated T cells. In some embodiments,the monitoring comprises determining the number of circulating tumorcells (CTC) in the individual. In some embodiments, the monitoringcomprises detecting a specific immune response against the plurality oftumor antigen peptides in the individual. In some embodiments, theplurality of tumor antigen peptides is adjusted based on the specificimmune response to provide a plurality of customized tumor antigenpeptides. In some embodiments, the method is repeated using theplurality of customized tumor antigen peptides.

In some embodiments, there is provided a method of cloning atumor-specific T cell receptor, comprising: (a) contacting a populationof PBMCs with a plurality of tumor antigen peptides (such as in thepresence of an immune checkpoint inhibitor) to obtain a population ofactivated PBMCs, (b) administering to an individual having a cancer aneffective amount of PBMCs; (c) isolating a T cell from the individual,wherein the T cell specifically recognizes a tumor antigen peptide inthe plurality of tumor antigen peptides; and (d) cloning a T cellreceptor from the T cell to provide the tumor-specific T cell receptor.In some embodiments, the activated PBMCs are administered for at leastthree times. In some embodiments, the interval between eachadministration of the activated PBMCs is about 2 weeks to about 5 months(such as about 3 months). In some embodiments, the activated PBMCs areadministered intravenously. In some embodiments, the population of PBMCsis obtained from the individual being treated. In some embodiments, theplurality of tumor antigen peptides comprises at least 10 tumor antigenpeptides. In some embodiments, the plurality of tumor antigen peptidescomprises a first core group of general tumor antigen peptides andoptionally a second group of cancer-type specific antigen peptides. Insome embodiments, the plurality of tumor antigen peptides comprises oneor more neoantigen peptides. In some embodiments, the method furthercomprises administering to the individual an effective amount of animmune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1,CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, theindividual is selected for the method of treating based on having a lowmutation load (such as in one or more MHC genes) in the cancer. In someembodiments, the individual is selected for the method of treating basedon having one or more (such as at least 5) neoantigens in theindividual. In some embodiments, the method further comprisesidentifying a neoantigen of the individual (such as by sequencing atumor sample from the individual), and incorporating a neoantigenpeptide in the plurality of tumor antigen peptides, wherein theneoantigen peptide comprises a neoepitope in the neoantigen. In someembodiments, the method further comprises monitoring the individualafter the administration of the activated PBMCs. In some embodiments,the monitoring comprises determining the number of circulating tumorcells (CTC) in the individual. In some embodiments, the monitoringcomprises detecting a specific immune response against the plurality oftumor antigen peptides in the individual. In some embodiments, theplurality of tumor antigen peptides is adjusted based on the specificimmune response to provide a plurality of customized tumor antigenpeptides. In some embodiments, the method is repeated using theplurality of customized tumor antigen peptides.

In some embodiments, the TCR is cloned from an individual that respondsto the MASCT method, for example, an individual having reduced CTCnumber or a low CTC number after the MASCT, an individual having aclinical evaluation of Stable Disease (SD), Complete Response (CR), orPartial Response (PR). In some embodiments, the TCR is cloned from anindividual that does not respond to the MASCT method. In someembodiments, the individual has a strong specific immune responseagainst the tumor antigen peptide. Specific immune response against anindividual tumor antigen peptide may be determined using any knownmethods in the art, for example, by measuring levels of cytotoxic factor(such as perforin or granzyme B), or cytokine release (such as IFNγ orTNFα) from T cells (or PBMCs) after stimulation by the individual tumorantigen peptide. An antibody-based assay, such as ELISPOT, may be usedto quantify the cytotoxic factor, or cytokine (such as IFNγ) levels. Insome embodiments, the cytokine (such as IFNγ) release level from T cells(or PBMCs) in response to a tumor antigen peptide is normalized to areference, such as a baseline cytokine release level, or a nonspecificcytokine release level of from T cells (or PBMCs) in response to anirrelevant peptide, to provide a cytokine (such as IFNγ) fold changevalue. In some embodiments, a cytokine (such as IFNγ) fold change valueof more than about any of 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, or morein an ELISPOT assay indicate strong specific immune response against thetumor antigen peptide. In some embodiments, the method of cloning a TCRfurther comprises determining the specific immune response of each ofthe plurality of tumor antigen peptides in the individual, such as in aPBMC sample of the individual.

The T cell may be isolated from a biological sample from the individualafter receiving the MASCT. In some embodiments, the biological sample isobtained from the individual after one cycle of MASCT. In someembodiments, the biological sample is obtained from the individual afterat least any of 2, 3, 4, 5, or more cycles of MASCT. In someembodiments, the biological sample is obtained from the individual afterat least about any of 1 week, 2 weeks, 3 weeks, 4 weeks 5 weeks, 6weeks, 2 months, or 3 months after receiving the MASCT. In someembodiments, the biological sample is obtained from the individual afterno more than about any of 6 months, 3 months, 2 months, 1 month, or lessafter receiving the MASCT. In some embodiments, the biological sample isa blood sample. In some embodiments, the biological sample is a PBMCsample. In some embodiments, the biological sample is a T cell sample.In some embodiments, the biological sample is a tumor sample containingCTLs. T cells may be isolated from the biological sample using any knownmethods in the art, for example, by flow cytometry or centrifugationmethods. In some embodiments, a plurality of T cells obtained from thebiological sample are screened for their specific immune responseagainst the plurality of tumor antigen peptides, for example, bystaining with multimers (such as pentamers or dextramers), or bydetermining the level of cytotoxic factor (such as perforin or granzymeB), or cytokine release (such as IFNγ or TNFα) by the cell.

The tumor antigen peptide that the T cell specifically recognizes can beany one from the tumor antigen peptide pool. In some embodiments, thetumor antigen peptide comprises an MHC-I restricted epitope. In someembodiments, the tumor antigen peptide comprises an MHC-II restrictedepitope. In some embodiments, the tumor antigen peptide is a generalcancer tumor antigen peptide. In some embodiments, the tumor antigenpeptide is a cancer-type specific tumor antigen peptide. In someembodiments, the tumor antigen peptide is a neoantigen peptide. In someembodiments, the tumor antigen peptide comprises an epitope derived fromCEA or hTERT.

TCRs can be cloned from T cells using any methods known in the art,including, but not limited to, PCR methods using primers thatspecifically annealing to known TCR variable domains. In someembodiments, amplicon rescued multiplex PCR (or arm-PCR) is used toclone the tumor-specific TCR. See, for example, U.S. Pat. No. 7,999,092.Methods for cloning tumor antigen-specific TCRs from single T cells mayalso be used to clone the TCR. See, for example, E. Kobayashi et al.Nature Medicine 19.11 (2013): 1542-1546. In some embodiments, the T cellis sequenced to determine the sequence of TCR genes, thereby allowingcloning of the TCR.

In some embodiments, the cloned TCRs are further incorporated in anexpression vector. In some embodiments, the cloned TCRs are furthertransduced (such as by a viral vector, or by physical or chemicalmethods) into a host cell (such as T cell) to express the TCR. In someembodiments, the host cell is a T cell. In some embodiments, the hostcell expressing the TCR is assayed for specific immune response to thetumor antigen peptide for validation. In some embodiments, the host cellis derived from a cell line. In some embodiments, the host cell is aprimary cell. In some embodiments, the host cell is a T cell. In someembodiments, the host cell is derived from a cancer patient.

Further provided herein are tumors-specific TCRs cloned using any of themethods described herein. In some embodiments, the tumor-specific TCR isfurther engineered to improve the physical/chemical properties and/orfunctions of the TCR. For example, the engineered tumor-specific TCR mayhave enhanced expression level, improved stability, enhanced bindingaffinity to the MHC-tumor-specific antigen peptide complexes, and/orenhanced signaling. In some embodiments, the tumor-specific TCRs areengineered based on the MHC subtype of the individual receivingimmunotherapy treatment using the tumor-specific TCRs. In someembodiments, the engineering comprises mutating one or more positions inthe variable regions of the cloned tumor-specific TCR. In someembodiments, the engineering comprises providing a fusion proteincomprising one or more domains or fragments of the cloned tumor-specificTCR.

In some embodiments, there is provided an isolated nucleic acid encodingthe tumor-specific TCR or components or derivatives thereof (such as theTCRα chain, the TCRβ chain, or the engineered tumor-specific TCR). Insome embodiments, there is provided an expression vector encoding thetumor-specific TCR or components or derivatives thereof (such as theTCRα chain, the TCRβ chain, or the engineered tumor-specific TCR). Insome embodiments, there is provided an isolated host cell expressing thetumor-specific TCR or components or derivatives thereof (such as theTCRα chain, the TCRβ chain, or the engineered tumor-specific TCR).

In some embodiments, there is provided an isolated T cell comprising thetumor-specific TCR or components or derivatives thereof (such as theTCRα chain, the TCRβ chain, or the engineered tumor-specific TCR). Insome embodiments, the endogenous TCR of the isolated T cell is knockedout. In some embodiments, the isolated T cell is a TCR-T cell. In someembodiments, there is provided a pharmaceutical composition comprisingthe isolated T cell and a pharmaceutically acceptable excipient. In someembodiments, the isolated T cell is derived from the individual havingthe cancer. In some embodiments, the isolated T cell is derived from theindividual to be treated with the isolated T cell or pharmaceuticalcomposition thereof.

The isolated T cells or pharmaceutical compositions thereof may beuseful for treating the individual from whom the tumor-specific TCR iscloned, or for treating another individual, such as an allogenicindividual, or an individual having the same MEW genotype and/orexpressing the same epitope on the cancer cells. In some embodiments,there is provided a method of treating a cancer in an individualcomprising administering to the individual an effective amount of any ofthe isolated T cells described herein or pharmaceutical compositionsthereof. The immunotherapy using the isolated T cell comprising thecloned tumor-specific TCR may be used singly or in combination withother treatments, such as immune checkpoint inhibitor, MASCT (includingPBMC-based MASCT and precision MASCT), chemotherapy, radiation, surgery,targeted therapy, etc., to achieve the desired clinical outcome.

Activated T Cells

The present invention further provides an isolated population of cellscomprising activated T cells, wherein less than about 1% of theactivated T cells are regulatory T (T_(REG)) cells. The isolatedpopulation of cells described herein may be prepared by any of themethod of preparing a population of activated T cells described in theprevious section. The isolated population of cells described herein isuseful for treating cancer, preventing tumor progression, or reducingimmune escape in an individual.

The isolated population of cells described herein comprise mainly ofactivated T cells. In some embodiments, at least about 90% of the cellsin the isolated population are activated T cells. In some embodiments,at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99% of thecells in the population are activated T cells. In some embodiments,about any of 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-98%, 50-70%,60-80%, 90-99%, 50-80%, 80-99%, 50-90%, 60-90%, 70-99%, or 50-99% of thecells in the isolated population are activated T cells.

In some embodiments, the isolated population of cells comprisesCD4⁺CD25⁺Foxp3⁺ cells. In some embodiments, the isolated population ofcells comprise less than about any of 10%, 5%, 3%, 1%, 0.9%, 0.8%, 0.7%,0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.01% CD4⁺CD25⁺Foxp3⁺ cells. Insome embodiments, the isolated population of cells comprise less thanabout any of 5-10%, 3-5%, 1-3%, 0.9-1%, 0.8-0.9%, 0.7-0.8%, 0.6-0.7%,0.5-0.6%, 0.4-0.5%, 0.3-0.4%, 0.2-0.3%, 0.1-0.2%, 0.1-0.5%, 0.5-1%,0.2-0.6%, 0.4-0.8%, 0.3-0.7%, or 0.3-0.5% CD4⁺CD25⁺Foxp3⁺ cells. In someembodiments, the isolated population of cells comprises about 0.3% toabout 0.5% CD4⁺CD25⁺Foxp3⁺ cells.

In some embodiments, the isolated population of cells comprisesregulatory T cells (T_(REG)). In some embodiments, the isolatedpopulation of cells comprise less than about any of 10%, 5%, 3%, 1%,0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.01% T_(REG)cells. In some embodiments, the isolated population of cells compriseless than about any of 5-10%, 3-5%, 1-3%, 0.9-1%, 0.8-0.9%, 0.7-0.8%,0.6-0.7%, 0.5-0.6%, 0.4-0.5%, 0.3-0.4%, 0.2-0.3%, 0.1-0.2%, 0.1-0.5%,0.5-1%, 0.2-0.6%, 0.4-0.8%, 0.3-0.7%, or 0.3-0.5% T_(REG) cells. In someembodiments, the isolated population of cells comprises about 0.3% toabout 0.5% T_(REG) cells.

In some embodiments, the isolated population of cells comprises CD3⁺CD8⁺cells. In some embodiments, the isolated population of cells comprisesabout any of 50%, 55%, 60%, 65%, 70%, 75%, or 80% CD3⁺CD8⁺ cells. Insome embodiments, the isolated population of cells comprise less thanabout any of 50-60%, 60-65%, 65-70%, 70-75%, 75-80%, 50-65%, 65-80%,65-70%, or 65-75% CD3⁺CD8⁺ cells. In some embodiments, the isolatedpopulation of cells comprises about 65% to about 75% CD3⁺CD8⁺ cells.

In some embodiments, the isolated population of cells comprisescytotoxic T cells. In some embodiments, the isolated population of cellscomprises about any of 50%, 55%, 60%, 65%, 70%, 75%, or 80% cytotoxic Tcells. In some embodiments, the isolated population of cells compriseless than about any of 50-60%, 60-65%, 65-70%, 70-75%, 75-80%, 50-65%,65-80%, 65-70%, or 65-75% cytotoxic T cells. In some embodiments, theisolated population of cells comprises about 65% to about 75% cytotoxicT cells.

In some embodiments, the isolated population of cells comprises CD3⁺CD4⁺cells. In some embodiments, the isolated population of cells compriseabout any of 10%, 13%, 16%, 18%, 20%, 22%, 25% or 30% CD3⁺CD4⁺ cells. Insome embodiments, the isolated population of cells comprise less thanabout any of 10-13%, 13-16%, 16-18%, 18-20%, 20-22%, 22-25%, 25-30%,16-20%, 18-22%, or 16-22% CD3⁺CD4⁺ cells. In some embodiments, theisolated population of cells comprises about 16% to about 22% CD3⁺CD4⁺cells.

In some embodiments, the isolated population of cells comprises helper Tcells. In some embodiments, the isolated population of cells compriseabout any of 10%, 13%, 16%, 18%, 20%, 22%, 25% or 30% helper T cells. Insome embodiments, the isolated population of cells comprise less thanabout any of 10-13%, 13-16%, 16-18%, 18-20%, 20-22%, 22-25%, 25-30%,16-20%, 18-22%, or 16-22% helper T cells. In some embodiments, theisolated population of cells comprises about 16% to about 22% helper Tcells.

In some embodiments, the isolated population of cells comprisesCD3⁺CD56⁺ cells. In some embodiments, the isolated population of cellscomprise about any of 10%, 12%, 13%, 13.5%, 14%, 14.5%, 15%, or 20%CD3⁺CD56⁺ cells. In some embodiments, the isolated population of cellscomprise less than about any of 10-12%, 12-13%, 13-13.5%, 13.5-14%,14-14.5%, 14.5-15%, 15-20%, 13-14%, 14-15%, 13.5-14.5%, or 13-15%CD3⁺CD56⁺ cells. In some embodiments, the isolated population of cellscomprises about 13% to about 15% CD3⁺CD56⁺ cells.

In some embodiments, the isolated population of cells comprises NaturalKiller (NK) T cells. In some embodiments, the isolated population ofcells comprise about any of 10%, 12%, 13%, 13.5%, 14%, 14.5%, 15%, or20% NK T cells. In some embodiments, the isolated population of cellscomprise less than about any of 10-12%, 12-13%, 13-13.5%, 13.5-14%,14-14.5%, 14.5-15%, 15-20%, 13-14%, 14-15%, 13.5-14.5%, or 13-15% NK Tcells. In some embodiments, the isolated population of cells comprisesabout 13% to about 15% NK T cells.

In some embodiments, the isolated population of cells comprises about0.3% to about 0.5% CD4⁺CD25⁺Foxp3⁺ cells, about 65% to about 75%CD3⁺CD8⁺ cells, and about 16% to about 22% CD3⁺CD4⁺ cells. In someembodiments, the isolated population of cells comprises about 0.3% toabout T_(REG) cells, about 65% to about 75% cytotoxic T cells, and about16% to about 22% helper T cells. In some embodiments, the isolatedpopulation of cells further comprises memory T cells.

In some embodiments, the activated T cells in any embodiment of theisolated population of cells are capable of eliciting specific immuneresponse to a plurality of tumor antigen peptides in vivo or ex vivo. Insome embodiments, the activated T cells are capable of increasingcytotoxic T cell activity in a human individual against more than onetumor antigen peptides. In some embodiments, the activated T cells arecharacterized by high expression or secretion level of pro-inflammatorysignal molecules, and low expression or secretion level ofimmunosuppressive cytokines. In some embodiments, the expression orsecretion level is determined by comparing the expression or secretionlevel of a molecule (such as a pro-inflammatory signal molecule, or animmunosuppressive cytokine) of the activated T cells to the controlexpression or secretion level. In some embodiments, the controlexpression or secretion level of a molecule is the expression orsecretion level of the molecule in a control population of T cellsmeasured under the same assay conditions. In some embodiments, thecontrol population of T cells is a population of T cells induced by aplurality of irrelevant peptides (such as peptides not corresponding toT cell receptor antigens, or random peptides). In some embodiments, thecontrol expression or secretion level of a molecule is an average ormedian expression or secretion level of the molecule in a plurality ofcontrol populations of T cells. In some embodiments, a high level ofexpression or secretion of a molecule in the activated T cells is atleast about any of 1.5, 2, 2.5, 3, 4, 5, 10, 20, 50, 100, 1000, or moretimes of the control expression or secretion level. In some embodiments,a low level of expression or secretion of a molecule in the activated Tcells is less than any of 0.001, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.75, or 0.8 times of the control expression or secretion level.

In some embodiments, the activated T cells express a plurality ofpro-inflammatory molecules, such as IFNγ, TNFα, granzyme B, perforin, orany combination thereof. In some embodiments, the activated T cells haveno or low expression of immunosuppressive cytokines, such as IL-10and/or IL-4. In some embodiments, the frequency of the activated T cells(such as CD3⁺CD4⁺ cells or CD3⁺CD8⁺cells) expressing immune-inhibitorymolecules, such as PD-1, is low. In some embodiments, the frequency ofthe activated T cells expressing PD-1 is less than about any of 10%, 5%,3%, 2%, or 1%. In some embodiment, less than about 5% of the activated Tcells express immune-inhibitory molecule PD-1.

The isolated population of cells described herein can be used togenerate specific immune memory in an individual when administered tothe individual. In some embodiments, the individual has memory T cellsthat can elicit specific T cell response against a plurality of tumorantigen peptides after about any of 2 weeks, 1 month, 2 months, 3months, 4 months, 6 months, 12 months, or more after administration ofthe isolated population of cells.

The isolated population of cells described herein can also be used toalter immune-inhibitory signals in vivo. In some embodiments, theisolated population of cells reduces immune-inhibitory molecule (such asPD-1) expression frequency on T cells (such as cytotoxic T cells orhelper T cells) in an individual when administered to the individual. Insome embodiments, the isolated population of cells reduces immunetolerance or immune escape of cancer cells in an individual.Accordingly, there is provided a method of reducing expression frequencyof an immune-inhibitory molecule, such as PD-1, in T cells of anindividual, comprising administering to the individual an effectiveamount of any embodiment of the isolated population of cells describedherein. Also provided herein is an immunotherapeutic compositioncomprising any embodiment of the isolated population of cells comprisingactivated T cells, and use of any embodiment of the isolated populationof cells in the manufacture of a medicament for treating a cancer in anindividual.

Compositions, Kits and Articles of Manufacture

The present invention further provides kits, compositions (such aspharmaceutical compositions), and commercial batches of the tumorantigen peptides for use in any embodiment of the MASCT method(including the PBMC-based MASCT method and precision MASCT) or the cell(such as antigen-loaded DCs, activated T cells, or activated PBMCs)preparation methods described herein.

In some embodiments, there is provided a kit useful for cancerimmunotherapy, comprising at least 10 tumor antigen peptides. In someembodiments, the kit comprises more than about any of 10, 15, 20, 25,30, 35, 40, 45, or 50 tumor antigen peptides. In some embodiments, theplurality of tumor antigen peptides comprises a first core group ofgeneral tumor antigen peptides. In some embodiments, the plurality oftumor antigen peptides comprises a first core group of general tumorantigen peptides and a second group of cancer-type specific antigenpeptides. In some embodiments, the first core group comprises about 10to about 20 general tumor antigen peptides. In some embodiments, thefirst core group comprises more than 1 general tumor antigen peptides.In some embodiments, the first core group comprises more than about anyof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 22, 25, 30, 40, or 50 general tumor antigen peptides. In someembodiments, the second group comprises about 1 to about 10 cancer-typespecific antigen peptides. In some embodiments, the second groupcomprises more than about any of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50 cancer-typespecific antigen peptides. In some embodiments, the second groupcomprises more than about any of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50 virus-specificantigen peptides. In some embodiments, the kit further comprises aboutany of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 22, 25, 30, 40, or 50 neoantigen peptides.

In some embodiments, there is provided a kit useful for cancerimmunotherapy, comprising at least 10 tumor antigen peptides, whereineach of the at least 10 tumor antigen peptides comprises at least oneepitope selected from the group consisting of SEQ ID NOs: 1-40. In someembodiments, there is provided a kit useful for cancer immunotherapy,comprising at least 10 tumor antigen peptides, wherein each of the atleast 10 tumor antigen peptides comprises at least one epitope selectedfrom the group consisting of SEQ ID NOs: 1-24. In some embodiments,there is provided a kit useful for cancer immunotherapy, comprising atleast 10 tumor antigen peptides selected from the group consisting ofthe tumor antigen peptides in FIG. 2B. In some embodiments, there isprovided a kit useful for cancer immunotherapy, comprising at least 10tumor antigen peptides comprising at least 10 tumor antigen peptidesselected from the group consisting of the tumor antigen peptides in FIG.2C and FIG. 29A. In some embodiments, there is provided a kit useful forcancer immunotherapy, comprising at least 10 tumor antigen peptidesderived from proteins selected from the group consisting of hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc,HBVp, CDCA1, KRAS, PARP4, MLL3, and MTHFR.

A person skilled in the art may use any combinations of tumor antigenpeptides from the first core group and optionally any combinations ofcancer-type specific antigen peptides from the second group, and/orneoantigen peptides to load a population of dendritic cells, which canfurther be used to prepare activated T cells useful for treating cancerin an individual. The kit may also be useful for PBMC-based MACTmethods, precision MASCT methods, or for cloning a tumor-specific TCRfrom an individual receiving the MASCT.

The kit may contain additional components, such as containers, reagents,culturing media, cytokines, buffers, antibodies, and the like tofacilitate execution of any embodiment of the MASCT method (includingthe PBMC-based MASCT method, and precision MASCT method), or methods forcloning a tumor-specific TCR from an individual receiving the MASCT. Forexample, in some embodiments, the kit further comprises a peripheralblood collection and storage apparatus, which can be used to collect anindividual's peripheral blood. In some embodiments, the kit furthercomprises containers and reagents for density gradient centrifugation ofperipheral blood, which can be used to isolate PBMCs from a sample ofhuman peripheral blood. In some embodiments, the kit further comprisesculturing media, cytokines, or buffers for obtaining dendritic cellsfrom peripheral blood. In some embodiments, the kit further comprisesculturing media, TLR agonists, reagents and buffers for loading thefirst core group and optionally the second group into dendritic cells toobtain dendritic cells loaded with a plurality of tumor antigenpeptides. In some embodiments, the kit further comprises cytokine,anti-CD3 antibody, buffers, immune checkpoint inhibitor, or culturingmedia for co-culturing T cells obtained from the peripheral blood withthe dendritic cells loaded with the plurality of tumor antigen peptides.In some embodiments, the kit further comprises reagents for determiningthe mutation load (such as in one or more MHC genes) in cancer cells. Insome embodiments, the kit further comprises an immune checkpointinhibitor for combination therapy with the MASCT. In some embodiments,the kit further comprises reagents for identifying a neoantigen (such asby sequencing) in a tumor sample. In some embodiments, the kit furthercomprises an ELISPOT assay for assessing specific immune responseagainst the plurality of tumor antigen peptides. In some embodiments,the kit further comprises reagents for cloning a tumor-specific TCR.

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., Mylar or plastic bags), and the like. Kits may optionally provideadditional components such as buffers and interpretative information.The present application thus also provides articles of manufacture,which include vials (such as sealed vials), bottles, jars, flexiblepackaging, and the like.

The instructions may also comprise instructions relating to the use ofthe tumor antigen peptides (and optionally additional componentsdescribed above). In some embodiments, the kit further comprises aninstructional manual, such as a manual describing a protocol of anembodiment of the MASCT methods (including the PBMC-based MASCT methodsand precision MASCT methods), an embodiment of the cell preparationmethods as described herein, or an embodiment of the methods of cloninga tumor-specific TCR. The instructions may also include information ondosage, dosing schedule, and route of administration of the antigenpresenting cells (such as dendritic cells), the activated T cells,and/or the activated PBMCs prepared using the kit for the intendedtreatment. In some embodiments, the kit further comprises instructionsfor selecting an individual for the MASCT method. In some embodiments,the kit further comprises instructions for determining the mutation loadof cancer cells, and/or determining the number of neoantigens in anindividual. In some embodiments, the kit further comprises instructionsfor administering an immune checkpoint inhibitor in combination with theMASCT, including, for example, information on dosage, dosing schedule,and route of administration of the immune checkpoint inhibitor. In someembodiments, the kit further comprises instructions for identifying aneoantigen (such as by sequencing) in a tumor sample. In someembodiments, the kit further comprises instructions for monitoring anindividual after receiving the MASCT. In some embodiments, the kitfurther comprises instructions for cloning a tumor-specific TCR.

The containers may be unit doses, bulk packages (e.g., multi-dosepackages) or sub-unit doses. For example, kits may be provided thatcontain sufficient tumor antigen peptides as disclosed herein to preparesufficient activated T cells and/or antigen-loaded dendritic cells (suchas dendritic cells) to provide effective treatment of an individual foran extended period, such as any of 3 weeks, 6 weeks, 9 weeks, 3 months,4 months, 5 months, 6 months, 8 months, 9 months, 1 year or more.

Kits may also include multiple unit doses of tumor antigen peptides andinstructions for use and packaged in quantities sufficient for storageand use in pharmacies, for example, hospital pharmacies and compoundingpharmacies.

In some embodiments, there is provided a commercial batch of thepopulation of tumor antigen peptides or the kit as described herein.“Commercial batch” used herein refers to a batch size that is at leastabout 10 mg. In some embodiments, the batch size is at least about anyof 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000,5000, or 10000 mg. In some embodiments, the commercial batch comprises aplurality of vials comprising any of the compositions (such as thepopulation of tumor antigen peptides or the kits) as described herein.In some embodiments, the commercial batch comprises at least about anyof 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 2000, 5000,or 10000 vials. For example, each vial contains at least about 0.1 mg oftumor antigen peptides. In some embodiments, the tumor antigen peptidesare in a liquid suspension. In some embodiments, the tumor antigenpeptides are in a powder form, such as a lyophilized powder.

Further provided are kits, compositions (such as pharmaceuticalcompositions), and commercial batches of any of the isolated populationof cells (such as dendritic cells, activated T cells, activated PBMCs,or isolated T cells comprising the tumor specific TCR) described herein.

The isolated population of cells described herein may be used inpharmaceutical compositions or formulations, by combining the isolatedpopulation of cells described with a pharmaceutically acceptablecarrier, excipients, stabilizing agents and/or other agents, which areknown in the art, for use in the methods of treatment, methods ofadministration, and dosage regimens described herein. In someembodiments, human albumin is used as a pharmaceutically acceptablecarrier.

Suitable pharmaceutical carriers include sterile water; saline,dextrose; dextrose in water or saline; condensation products of castoroil and ethylene oxide combining about 30 to about 35 moles of ethyleneoxide per mole of castor oil; liquid acid; lower alkanols; oils such ascorn oil; peanut oil, sesame oil and the like, with emulsifiers such asmono- or di-glyceride of a fatty acid, or a phosphatide, e.g., lecithin,and the like; glycols; polyalkylene glycols; aqueous media in thepresence of a suspending agent, for example, sodiumcarboxymethylcellulose; sodium alginate; poly(vinylpyrolidone); and thelike, alone, or with suitable dispensing agents such as lecithin;polyoxyethylene stearate; and the like. The carrier may also containadjuvants such as preserving stabilizing, wetting, emulsifying agentsand the like together with the penetration enhancer. The final form maybe sterile and may also be able to pass readily through an injectiondevice such as a hollow needle. The proper viscosity may be achieved andmaintained by the proper choice of solvents or excipients.

The pharmaceutical compositions described herein may include otheragents, excipients, or stabilizers to improve properties of thecomposition. Examples of suitable excipients and diluents include, butare not limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate and mineral oil. Insome embodiments, the pharmaceutical composition is formulated to have apH in the range of about 4.5 to about 9.0, including for example pHranges of about any one of 5.0 to about 8.0, about 6.5 to about 7.5, orabout 6.5 to about 7.0. In some embodiments, the pharmaceuticalcomposition can also be made to be isotonic with blood by the additionof a suitable tonicity modifier, such as glycerol.

In some embodiments, the isolated cell compositions (such aspharmaceutical compositions) is suitable for administration to a human.In some embodiments, the compositions (such as pharmaceuticalcompositions) is suitable for administration to a human by parenteraladministration. Formulations suitable for parenteral administrationinclude aqueous and non-aqueous, isotonic sterile injection solutions,which can contain anti-oxidants, buffers, bacteriostats, and solutesthat render the formulation compatible with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizingagents, and preservatives. The formulations can be presented inunit-dose or multi-dose sealed containers, such as ampules and vials,and can be stored in a condition requiring only the addition of thesterile liquid excipient methods of treatment, methods ofadministration, and dosage regimens described herein (i.e., water) forinjection, immediately prior to use. In some embodiments, thecompositions (such as pharmaceutical compositions) is contained in asingle-use vial, such as a single-use sealed vial. In some embodiments,each single-use vial contains about 10⁹ activated T cells. In someembodiments, each single-use vial contains enough activated T cells tobe expanded to about 10⁹ activated T cells. In some embodiments, thecompositions (such as pharmaceutical compositions) is contained in amulti-use vial. In some embodiments, the compositions (such aspharmaceutical compositions) is contained in bulk in a container.

Also provided are unit dosage forms comprising the isolated cellcompositions (such as pharmaceutical compositions) and formulationsdescribed herein. These unit dosage forms can be stored in a suitablepackaging in single or multiple unit dosages and may also be furthersterilized and sealed. In some embodiments, the composition (such aspharmaceutical composition) also includes one or more other compounds(or pharmaceutically acceptable salts thereof) that are useful fortreating cancer. In various variations, the number of activated T cellsin the pharmaceutical composition is included in any one of thefollowing ranges: about 1×10⁸ to about 5×10⁸, about 5×10⁸ to about9×10⁸, about 9×10⁸ to about 1×10⁹, about 1×10⁹ to about 2×10⁹, about2×10⁹ to about 3×10⁹, about 3×10⁹ to about 4×10⁹, about 4×10⁹ to about5×10⁹, about 5×10⁹ to about 6×10⁹, about 6×10⁹ to about 1×10¹⁰, about1×10⁹ to about 3×10⁹, about 3×10⁹ to about 5×10⁹, about 5×10⁹ to about7×10⁹, about 7×10⁹ to about 1×10¹⁰, about 1×10⁹ to about 5×10⁹, about5×10⁹ to about 1×10¹⁰, about 3×10⁹ to about 7×10⁹, about 1×10¹⁰ to about1.5×10¹⁰, about 1×10¹⁰ to about 2×10¹⁰, or about 1×10⁹ to about 1×10¹⁰cells. In some embodiments, the activated T cells are the onlypharmaceutically active agent for the treatment of cancer that iscontained in the composition.

In some embodiments, there is provided a dosage form (e.g., a unitdosage form) for the treatment of cancer comprising any one of theisolated cell compositions (such as pharmaceutical compositions)described herein. In some embodiments, there are provided articles ofmanufacture comprising the compositions (such as pharmaceuticalcompositions), formulations, and unit dosages described herein insuitable packaging for use in the methods of treatment, methods ofadministration, and dosage regimens described herein. Suitable packagingfor compositions (such as pharmaceutical compositions) described hereinare known in the art, and include, for example, vials (such as sealedvials), vessels (such as sealed vessels), ampules, bottles, jars,flexible packaging (e.g., sealed Mylar or plastic bags), and the like.These articles of manufacture may further be sterilized and/or sealed.

The present application further provides kits comprising any of theisolated population of cells, compositions (such as pharmaceuticalcompositions), formulations, unit dosages, and articles of manufacturedescribed herein for use in the methods of treatment, methods ofadministration, and dosage regimens described herein. Kits describedherein include one or more containers comprising the activated T cells.

In some embodiments, there is provided a commercial batch of activated Tcells described herein. “Commercial batch” used herein refers to a batchsize that is at least about 1×10⁹ activated T cells. In someembodiments, the batch size is at least about any of 1×10⁹, 2×10⁹,3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 2×10¹⁰, 5×10¹⁰,or 1×10¹¹ cells. In some embodiments, the commercial batch comprises aplurality of vials comprising any of the compositions (such aspharmaceutical compositions) described herein. In some embodiments, thecommercial batch comprises at least about any of 5, 10, 15, 20, 25, 50,75, or 100 vials. For example, each vial contains at least about 1×10⁹activated T cells.

The examples and exemplary embodiments below are intended to be purelyexemplary of the invention and should therefore not be considered tolimit the invention in any way. The following examples and detaileddescription are offered by way of illustration and not by way oflimitation.

EXEMPLARY EMBODIMENTS

Embodiment 1. In some embodiments, there is provided a method oftreating a cancer in an individual, comprising administering to theindividual an effective amount of activated T cells, wherein theactivated T cells are prepared by co-culturing a population of T cellswith a population of dendritic cells loaded with a plurality of tumorantigen peptides.

Embodiment 2. In some further embodiments of embodiment 1, theindividual has previously been administered with an effective amount ofdendritic cells loaded with the plurality of tumor antigen peptides.

Embodiment 3. In some further embodiments of embodiment 1, the methodfurther comprises administering to the individual an effective amount ofthe dendritic cells loaded with the plurality of tumor antigen peptides.

Embodiment 4. In some further embodiments of embodiment 3, the dendriticcells are administered prior to the administration of the activated Tcells.

Embodiment 5. In some further embodiments of embodiment 4, the dendriticcells are administered about 7 days to about 21 days (such as about 7days to about 14 days, or about 14 days to about 21 days) prior to theadministration of the activated T cells.

Embodiment 6. In some further embodiments of any one of embodiments 1-5,the method further comprises preparing the activated T cells byco-culturing the population of T cells with the population of dendriticcells loaded with the plurality of tumor antigen peptides.

Embodiment 7. In some further embodiments of embodiment 6, thepopulation of T cells is co-cultured with the population of dendriticcells for about 7 days to about 21 days (such as about 7 days to about14 days, or about 14 days to about 21 days).

Embodiment 8. In some further embodiments of any one of embodiments 1-7,the population of T cells is contacted with an immune checkpointinhibitor prior to the co-culturing.

Embodiment 9. In some further embodiments of any one of embodiments 1-8,the population of T cells is co-cultured with the population ofdendritic cells loaded with the plurality of tumor antigen peptides inthe presence of an immune checkpoint inhibitor.

Embodiment 10. In some further embodiments of embodiment 8 or embodiment9, the immune checkpoint inhibitor is an inhibitor of an immunecheckpoint molecule selected from the group consisting of PD-1, PD-L1,and CTLA-4.

Embodiment 11. In some further embodiments of any one of embodiments1-10, the method further comprises preparing the population of dendriticcells loaded with the plurality of tumor antigen peptides.

Embodiment 12. In some further embodiments of embodiment 11, thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides is prepared by contacting a population of dendritic cells withthe plurality of tumor antigen peptides.

Embodiment 13. In some further embodiments of embodiment 12, thepopulation of dendritic cells loaded with the plurality of tumor antigenpeptides is prepared by contacting the population of dendritic cellswith the plurality of tumor antigen peptides in the presence of acomposition that facilitates the uptake of the plurality of tumorantigen peptides by the dendritic cells.

Embodiment 14. In some further embodiments of any one of embodiments1-13, the population of T cells and the population of dendritic cellsare derived from the same individual.

Embodiment 15. In some further embodiments of embodiment 14, thepopulation of T cells and the population of dendritic cells are derivedfrom the individual being treated.

Embodiment 16. In some embodiments, there is provided a method ofpreparing a population of activated T cells, wherein the methodcomprises: a) inducing differentiation of the population of monocytesinto a population of dendritic cells; b) contacting the population ofdendritic cells with a plurality of tumor antigen peptides to obtain apopulation of dendritic cells loaded with the plurality of tumor antigenpeptides; and c) co-culturing the population of dendritic cells loadedwith the plurality of tumor antigen peptides and the population ofnon-adherent PBMCs to obtain the population of activated T cells,wherein the population of monocytes and the population of non-adherentPBMCs are obtained from a population of PBMCs from an individual.

Embodiment 17. In some further embodiments of embodiment 16, step b)comprises contacting the population of dendritic cells with theplurality of tumor antigen peptides in the presence of a compositionthat facilitates the uptake of the plurality of tumor antigen peptidesby the dendritic cells.

Embodiment 18. In some further embodiments of embodiment 16 orembodiment 17, step d) further comprises contacting the population ofdendritic cells loaded with the plurality of tumor antigen peptides witha plurality of Toll-like Receptor (TLR) agonists to induce maturation ofthe population of dendritic cells loaded with the plurality of tumorantigen peptides.

Embodiment 19. In some further embodiments of any one of embodiments16-18, step f) further comprises contacting the population of activatedT cells with a plurality of cytokines to induce proliferation anddifferentiation of the population of activated T cells.

Embodiment 20. In some further embodiments of embodiment 19, theplurality of cytokines comprises IL-2, IL-7, IL-15 or IL-21.

Embodiment 21. In some further embodiments of any one of embodiments16-20, the population of non-adherent PBMCs is contacted with an immunecheckpoint inhibitor prior to the co-culturing.

Embodiment 22. In some further embodiments of any one of embodiments16-21, step c) comprises co-culturing the population of dendritic cellsloaded with the plurality of tumor antigen peptides and the populationof non-adherent PBMCs in the presence of an immune checkpoint inhibitor.

Embodiment 23. In some further embodiments of embodiment 21 orembodiment 22, the immune checkpoint inhibitor is an inhibitor of animmune checkpoint molecule selected from the group consisting of PD-1,PD-L1, and CTLA-4.

Embodiment 24. In some embodiments, there is provided a method oftreating a cancer in an individual, comprising administering to theindividual an effective amount of a population of activated T cellsprepared by the method of any one of the methods described inembodiments 16-23.

Embodiment 25. In some further embodiments of embodiments 24, thepopulation of PBMCs is obtained from the individual being treated.

Embodiment 26. In some further embodiments of any one of embodiments1-15 and 24-25, the activated T cells are administered to the individualfor at least three times.

Embodiment 27. In some further embodiments of embodiment 26, intervalbetween each administration of the activated T cells is about 0.5 monthto about 5 months.

Embodiment 28. In some further embodiments of any one of embodiments1-15 and 24-27, the activated T cells are administered intravenously.

Embodiment 29. In some further embodiments of any one of embodiments1-15 and 24-28, the activated T cells are administered at a dose of atleast about 3×10⁹ cells/individual.

Embodiment 30. In some further embodiments of embodiment 29, theactivated T cells are administered at about 1×10⁹ to about 1×10¹⁰cells/individual.

Embodiment 31. In some further embodiments of any one of embodiments2-15 and 24-30, the dendritic cells loaded with the plurality of tumorantigen peptides are administered for at least three times.

Embodiment 32. In some further embodiments of embodiment 31, theinterval between each administration of the dendritic cells is about 0.5month to about 5 months.

Embodiments 33. In some further embodiments of any one of embodiments2-15 and 24-32, the dendritic cells loaded with the plurality of tumorantigen peptides are administered subcutaneously.

Embodiment 34. In some further embodiments of any one of embodiments2-15 and 24-33, the dendritic cells are administered at a dose of about1×10⁶ to about 5×10⁶ cells/individual.

Embodiment 35. In some embodiments, there is provided a method oftreating a cancer in an individual, comprising: a) contacting apopulation of PBMCs with a plurality of tumor antigen peptides to obtaina population of activated PBMCs, and b) administering to the individualan effective amount of the activated PBMCs.

Embodiment 36. In some further embodiments of embodiment 35, step (a)comprises contacting the population of PBMCs with a plurality of tumorantigen peptides in the presence of an immune checkpoint inhibitor

Embodiment 37. In some further embodiments of embodiment 36, the immunecheckpoint inhibitor is an inhibitor of an immune checkpoint moleculeselected from the group consisting of PD-1, PD-L1, and CTLA-4.

Embodiment 38. In some further embodiments of any one of embodiments35-37, the activated PBMCs are administered for at least three times.

Embodiment 39. In some further embodiments of embodiment 38, theinterval between each administration of the activated PBMCs is about 0.5month to about 5 months.

Embodiment 40. In some further embodiments of any one of embodiments35-39, the activated PBMCs are administered intravenously.

Embodiment 41. In some further embodiments of any one of embodiments35-40, the activated PBMCs are administered at a dose of about 1×10⁹ toabout 1×10¹⁰ cells/individual.

Embodiment 42. In some further embodiments of any one of embodiments1-41, the plurality of tumor antigen peptides is each about 20 to about40 amino acids long.

Embodiment 43. In some further embodiments of any one of embodiments1-42, the plurality of tumor antigen peptides comprises at least onepeptide comprising an MHC-I epitope.

Embodiment 44. In some further embodiments of any one of embodiments1-43, the plurality of tumor antigen peptides comprises at least onepeptide comprising an MHC-II epitope.

Embodiment 45. In some further embodiments of embodiment 43 orembodiment 44, the at least one peptide comprising an MHC-I epitope orMHC-II epitope further comprise additional amino acids flanking theepitope at the N-terminus, the C-terminus, or both.

Embodiment 46. In some further embodiments of any one of embodiments1-45, the plurality of tumor antigen peptides comprises a first coregroup of general tumor antigen peptides.

Embodiment 47. In some further embodiments of embodiment 46, theplurality of tumor antigen peptides further comprises a second group ofcancer-type specific antigen peptides.

Embodiment 48. In some further embodiments of any one of embodiment 46or embodiment 47, the first core group comprises about 10 to about 20general tumor antigen peptides.

Embodiment 49. In some further embodiments of embodiment 47 orembodiment 48, the second group comprises about 1 to about 10cancer-type specific antigen peptides.

Embodiment 50. In some further embodiments of any one of embodiments1-49, the plurality of tumor antigen peptides comprises a neoantigenpeptide.

Embodiment 51. In some further embodiments of embodiment 50, theneoantigen peptide is selected based on the genetic profile of a tumorsample from the individual.

Embodiment 52. In some further embodiments of any one of embodiments1-15 and 24-51, the cancer is selected from the group consisting ofhepatic cellular carcinoma, cervical cancer, lung cancer, colorectalcancer, lymphoma, renal cancer, breast cancer, pancreatic cancer,gastric cancer, esophageal cancer, ovarian cancer, prostate cancer,nasopharyngeal cancer, melanoma and brain cancer.

Embodiment 53. In some further embodiments of any one of embodiments1-15 and 24-52, the method further comprises administering to theindividual an effective amount of an immune checkpoint inhibitor.

Embodiment 54. In some further embodiments of embodiment 53, the immunecheckpoint inhibitor is an inhibitor of an immune checkpoint moleculeselected from the group consisting of PD-1, PD-L1, and CTLA-4.

Embodiment 55. In some further embodiments of any one of embodiments1-15 and 24-54, the individual is selected for the method of treatingbased on the mutation load in the cancer.

Embodiment 56. In some further embodiments of any one of embodiments1-15 and 24-55, the individual has a low mutation load in the cancer.

Embodiment 57. In some further embodiments of embodiment 56, theindividual has a low mutation load in one or more MHC genes.

Embodiment 58. In some further embodiments of embodiment 57, theindividual has no more than about 10 mutations in the one or more MHCgenes.

Embodiment 59. In some further embodiments of embodiment 57 orembodiment 58, the individual has no mutation in B2M.

Embodiment 60. In some further embodiments of any one of embodiments57-59, wherein the individual has no mutation in the functional regionsof the one or more MHC genes.

Embodiment 61. In some further embodiments of any one of embodiments55-60, the mutation load of the cancer is determined by sequencing atumor sample from the individual.

Embodiment 62. In some further embodiments of any one of embodiments1-15 and 24-61, the individual is selected for the method of treatingbased on having one or more neoantigens in the cancer.

Embodiment 63. In some further embodiments of any one of embodiments1-15 and 24-62, the individual has at least 5 neoantigens.

Embodiment 64. In some further embodiments of embodiment 62 orembodiment 63, the method further comprises identifying a neoantigen ofthe cancer, and incorporating a neoantigen peptide in the plurality oftumor antigen peptides, wherein the neoantigen peptide comprises aneoepitope in the neoantigen.

Embodiment 65. In some further embodiments of any one of embodiments62-64, the neoantigen is identified by sequencing a tumor sample fromthe individual.

Embodiment 66. In some further embodiments of embodiment 65, saidsequencing is targeted sequencing of cancer-associated genes.

Embodiment 67. In some further embodiments of any one of embodiments64-66, the method further comprises determining the affinity of theneoepitope to an MHC molecule.

Embodiment 68. In some further embodiments of any one of embodiments64-67, the method further comprises determining the affinity of thecomplex comprising the neoepitope and an MHC molecule to a T cellreceptor.

Embodiment 69. In some further embodiments of embodiment 67 orembodiment 68, the MHC molecule is an MHC class I molecule.

Embodiment 70. In some further embodiments of any one of embodiments67-69, the MHC molecule is from the individual.

Embodiment 71. In some further embodiments of any one of embodiments1-15 and 24-70, the method further comprises monitoring the individualafter the administration of the activated T cells or the activatedPBMCs.

Embodiment 72. In some further embodiments of embodiment 71, themonitoring comprises determining the number of circulating tumor cells(CTC) in the individual.

Embodiment 73. In some further embodiments of embodiment 71 orembodiment 72, the monitoring comprises detecting a specific immuneresponse against the plurality of tumor antigen peptides in theindividual.

Embodiment 74. In some further embodiments of embodiment 73, theplurality of tumor antigen peptides is adjusted based on the specificimmune response to provide a plurality of customized tumor antigenpeptides.

Embodiment 75. In some further embodiments of embodiment 74, the methodof treating is repeated using the plurality of customized tumor antigenpeptides.

Embodiment 76. In some further embodiments of any one of embodiments1-15 and 24-75, the individual is a human individual.

Embodiment 77. In some embodiments, there is provided a method ofcloning a tumor-specific T cell receptor, comprising: (a) treating anindividual with the method of any one of embodiments 1-15 and 24-76; (b)isolating a T cell from the individual, wherein the T cell specificallyrecognizes a tumor antigen peptide in the plurality of tumor antigenpeptides; and (c) cloning a T cell receptor from the T cell to providethe tumor-specific T cell receptor.

Embodiment 78. In some further embodiments of embodiment 77, theindividual has a strong specific immune response against the tumorantigen peptide.

Embodiment 79. In some further embodiments of embodiment 77 orembodiment 78, the T cell is isolated from a PBMC sample of theindividual.

Embodiment 80. In some further embodiments of any one of embodiments77-79, the tumor antigen peptide is a neoantigen peptide.

Embodiment 81. In some embodiments, there is provided a tumor-specific Tcell receptor cloned using the method of any one of embodiments 77-80.

Embodiment 82. In some embodiments, there is provided an isolated T cellcomprising the tumor-specific T cell receptor of embodiment 81.

Embodiment 83. In some embodiments, there is provided a method oftreating a cancer in an individual comprising administering to theindividual an effective amount of the isolated T cell of embodiment 82.

Embodiment 84. In some embodiments, there is provided an isolatedpopulation of cells prepared by the method of any one of embodiments16-23 and 42-51.

Embodiment 85. In some embodiments, there is provided an isolatedpopulation of cells comprising activated T cells, wherein less thanabout 1% of the activated T cells are regulatory T (T_(REG)) cells.

Embodiment 86. In some further embodiments of embodiment 84 orembodiment 85, the isolated population of cells comprises about 0.3% toabout 0.5% CD4⁺CD25⁺Foxp3⁺ cells.

Embodiment 87. In some further embodiments of any one of embodiments84-86, the isolated population of cells comprises about 65% to about 75%CD3⁺CD8⁺ cells.

Embodiment 88. In some further embodiments of any one of embodiments84-87, the isolated population of cells comprises about 16% to about 22%of CD3⁺CD4⁺ cells.

Embodiment 89. In some further embodiments of any one of embodiments84-88, the isolated population of cells comprises about 13% to about 15%CD3⁺CD56⁺ cells.

Embodiment 90. In some further embodiments of any one of embodiments84-89, the activated T cells are capable of eliciting specific responseto a plurality of tumor antigen peptides in vivo or ex vivo.

Embodiment 91. In some further embodiments of embodiment 90, theactivated T cells express a plurality of pro-inflammatory molecules.

Embodiment 92. In some further embodiments of embodiment 91, theplurality of pro-inflammatory molecules comprises IFNγ, TNFα, granzymeB, or perforin.

Embodiment 93. In some further embodiments of any one of embodiments84-92, the activated T cells have no or low expression of a plurality ofimmunosuppressive cytokines.

Embodiment 94. In some further embodiments of embodiment 93, theplurality of immunosuppressive cytokines comprises IL-10 or IL-4.

Embodiment 95. In some further embodiments of any one of embodiments84-94, less than about 5% of the activated T cells expressimmune-inhibitory molecule PD-1.

Embodiment 96. In some further embodiments of any one of embodiments84-95, at least about 90% of the cells in the isolated population ofcells are activated T cells.

Embodiment 97. In some embodiments, there is provided a compositioncomprising at least 10 tumor antigen peptides, wherein each of the atleast 10 tumor antigen peptides comprises at least one epitope selectedfrom the group consisting of SEQ ID NOs: 1-40.

Embodiment 98. In some further embodiments of embodiment 56, the atleast 10 tumor antigen peptides each comprises one or more epitopesencoded by a cancer-associated gene selected from the group consistingof hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR,AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

EXAMPLES

The examples described herein are not intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used (forexample, amounts, temperature, etc.), but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1—A Clinical and Mechanistic Study of MASCT in Treating HCCIntroduction

Hepatocellular carcinoma (HCC) is one of cancers with high mortality,and frequently occurs in the Chinese patients with chronic hepatitis Bvirus (HBV) infection. Although current standard of cares (SOC), such asresection, liver transplantation, transcatheter arterialchemoembolization (TACE), may improve the survival of patients, HCC israrely cured and has a high risk of recurrence and metastasis. Here, weapplied the MASCT method to a group of Chinese patients suffering fromHCC and co-infected with HBV.

Autologous T celled activated by multiple tumor antigens were preparedex vivo from patients' PBMC according to an embodiment of the MASCTmethod, and administered to the patients by infusion. With the MASCTstrategy, we selectively activated and amplified tumor antigen-specificT cells from the autologous T cell repertoires of the patients withrelevant tumor antigen peptides. This ensures that the resulting cellswould specifically recognize tumor cells without cross-reactivityagainst healthy tissue, since these T cells have survived from centralselection in the thymus, where all the strong self-reacting T cells thatare harmful to the hosts have been removed. We found that the activatedT cells led to the expansion of HCC-specific T cells including botheffecter and memory T cells in vivo and improved the progression freeoutcome in patients with HCC.

Results

The Manufacturing Process and Characteristics of MASCT

The cell manufacturing process of MASCT cell therapy is shown in FIG.2A. Briefly, the immature dendritic cells (iDCs), differentiated frommonocytes isolated from patients' PBMC, were pulsed with tumor antigenpeptides pool to become mature DCs (mDCs) under the help of TLRagonists. The autologous T cells from the same source of PBMCs weremaintained in cytokines and followed co-culturing with mDCs prepared asdescribed above for another 7-10 days. The antigen peptide poolconsisted of multiple tumor associate antigens which were overexpressedin cancerous hepatocytes of the patients with HCC. Some of them werealready used in clinical trials (FIG. 2B)(1-15). The HCC antigenpeptides pool included ten tumor-associated antigens (TAAs), such assurvivin, NY-ESO-1, carcino-embryonic antigen (CEA), and so on, whichwere overexpressed in numerous kinds of tumor cells including HCC; andtwo HCC specific antigens, named alpha fetoprotein (AFP) and glypican-3(GPC3), which were commonly expressed in cancerous hepatocytes of thepatients with HCC. Two of the HBV associated antigens named HBV coreantigen and HBV DNA polymerase, were also included in the peptides pool,since HCC patients in China were mostly correlated with chronichepatitis B virus (HBV) infection. Moreover, each of the peptides wassynthesized with a length of 20-40 amino acids and containing severalpreviously identified T cell recognition epitopes (FIG. 2C) for bothclass I and class II HLA molecules. The peptides were chemicallysynthesized under GMP condition.

Experiments showed that peptides were effectively internalized by iDCsand primarily localized in the cytosol (FIG. 3), which wouldconsequently promote the cross-presentation by MHC I molecules. Afterstimulation with toll-like receptor (TLR) ligands, mDCs demonstratedfull immune functional properties, especially for the high levelsecretion of IL12 (FIGS. 4 A-B).

After co-culturing, the resulting T cells had proliferated for at least45-times greater, from median, 8.9×10⁷ cells (range, 5×10⁷˜1.6×10⁸cells) to median, 6.2×10⁹ cells (range, 4.1×10⁹˜9×10⁹ cells; FIG. 5A).The resulting cells were almost exclusively CD3⁺ T cells (95%±1%) with amajor phenotype of CD3⁺CD8⁺ (70%±5%) and small part of CD3⁺CD4⁺ (19%±3%)and CD3⁺CD56⁺ (14%±1%), but nearly no CD4⁺CD25⁺Foxp3⁺ regulatory T cells(T_(REG), 0.4%±0.1%) (FIG. 5B).

The Immunological Function of the Resulting T Cells

The subsets analysis of the resulting T cells revealed a poly-functionalproperty in the major subset of CD3⁺CD8⁺ cytotoxic T cells, as well asin the minor subsets of CD3⁺CD4⁺ helper T cells and CD3⁺CD56⁺ NKT cells(16), characterized by the co-expression of IFNγ, TNFα and granzyme B(FIG. 5C, D), as compared to the non-activated T cells isolated frompatients (FIG. 5E). The pro-inflammatory cytokines such as IFNγ and TNFαwere also found in supernatants of the resulting cells, but barelyimmunosuppressive cytokines such as IL10 and IL4 were detected (FIG.6A). Others (17, 18) and we have detected a higher frequency of bothCD3⁺CD8⁺ and CD3⁺CD4⁺ subsets of T cells expressing immune inhibitorymolecule PD-1 on their surface from HCC patients compared to healthydonors, suggesting a T cells exhaustion in HCC patients (FIG. 6B-C).However, this immune tolerance status of T cells could be significantlyreversed in both CD3⁺CD8⁺ T cell subset (n=7, p=0.02) and CD3⁺CD4⁺ Tcell subset (n=7, p<0.01) by the stimulation of DCs pulsed with multipletumor antigens (FIG. 6D-E). Moreover, the activated T cells generatedfrom HLA-A2⁺ patients exhibited superior cytotoxic activity against theHLA-A2⁺ HCC cell line HepG2 than the HLA-A2⁻Huh-7 cells suggesting aHLA-restricted killing. While the resulting T cells generated fromHLA-A2⁻ patients (n=7) exhibited similar cytotoxic activity against boththe HLA-A2⁺ and the HLA-A2⁻ HCC cell lines, which may contribute to thenon-specific killing performed by CD3⁺CD56⁺ NKT cells (FIG. 6F).

MASCT-Induced Antigen Specific Immune Responses in HCC Patients

To investigate whether MASCT treatment could bring an improvement of theimmune environment in HCC patients, we measured the frequency of T_(REG)in patents' PBMCs and found a significant down-regulation of these cellsafter three applications of MASCT (FIG. 11A). We also detected theproliferation (FIG. 11B) and IFNγ production (FIG. 11C) of specific Tcells against tumor antigens in the patients' PBMCs after stimulationwith peptides pool compared to stimulation with irrelevant peptides. Andwe have observed a significantly increased response in HCC patients'PBMCs (n=6) after stimulation with HCC-antigen peptides pool, ascompared to PBMCs stimulated with irrelevant peptides (Antigen specificproliferation: p=0.027; antigen specific IFNγ production: p=0.024, FIGS.11B and 11C). The IFNγ producing HCC specific CD8+ T cells alsoco-expressed CD27 and CD28 on their surfaces (FIG. 11D), suggesting ahigh potential to acquire immune memory phenotype(19, 20). Moreover, toinvestigate whether the HCC antigen-specific T cell proliferation andIFNγ production were induced or enhanced during the MASCT treatment, wecompared these immune responses of patients' PBMCs before and after 3times treatment of MASCT. The results show that these HCC specific Tcell proliferation and IFNγ production were robust, and graduallyaccumulative immune responses were detected in patients after multipletreatment of MASCT (FIGS. 11E, and 11F). Thus, in MASCT treatedpatients, both down-regulation of T_(REG) and up-regulation oftumor-specific T cells were detected, demonstrating the improvement ofimmune environment in HCC patients after MASCT treatment.

The MASCT-Induced Immune Responses Against Each Antigen Peptide in thePool

To further examine the specific response against each kind of tumorantigen peptide out of the pool, PBMCs from 6 HCC patients (all of themwere HBV⁺) after multiple treatments of MASCT cell therapy were isolatedand stimulated with individual antigen peptides. The production of IFNγwas measured by ELISPOT assay. The specific responses against tumorantigen peptides were clearly raised in all of the 6 patients (HBV⁺),whereas the specific immune responding pattern against each antigenpeptides was distinct. Most patients responded to CEA (5/6), HBV coreantigen (5/6) survivin (4/6), VEGFR (4/6), AFP (4/6), GPC3 (4/6) and HBVDNA polymerase (4/6). But fewer patients responded to p53 (2/6), CCDN1(2/6) and MET (1/6) (FIG. 12A), which may due to the diversity ofantigen expression in tumors and variability of immune environments indifferent patients. However, very few immune responses against thesetumor antigens were observed in patients (n=5) without MASCT celltherapy (FIG. 12B). Moreover, we have longitudinally studied the dynamicchanges of immune responses in 2 patients with HCC (B stage) duringtheir treatments of MASCT cell therapy and discovered that the specificimmune responses against tumor antigen peptides were graduallyincreased, and the immune responses patterns for the 2 patients weredifferent (FIGS. 12C, 12D). We were able to detect tumor antigenspecific responses of T cells in patients more than 4 months after thelast MASCT treatment (data now shown), suggesting establishment oflong-term immune response by memory T cells. Both the raised level oftumor antigens specific T cells and the decreased level of T_(REG) maylead to a better clinical outcome for patients.

Interim Analysis of Clinical Benefits of MASCT

To investigate the clinical benefits of MASCT cell therapy, we haveretrospectively studied the cancer progressive circumstances of HCCpatients. From 2012 up to present, 100 patients were diagnosed as Bstage of HCC (FIG. 7A). Out of all patients, 33 were further analyzed,since they were continuously treated for at least 1 year. 15 of themhave only received conventional treatments for HCC such as resection,TACE and RFA (FIG. 8A). The other 16 patients have received MASCT celltherapies in addition to the standard of care. Among them, 13 patientshave received repeat MASCT cell therapies 3) every 1-3 months combinedwith conventional treatments (FIG. 9A); 3 patients who only had a singleMASCT cell therapy were not further analyzed, since they did not followthe protocol of repeating treatments. During each treatment, 2-5×10⁷cells/kg body weight (or at least 1×10⁹ per person) were infused. Noclear toxicity was observed. If the tumors were detected as recurrence,growth or metastasis by Computed Tomography (CT) scan, the patients wereevaluated as PD (progressive disease). The time and received treatmentsfrom diagnosis to disease progression were indicated. If the tumors werenot progressive, the evaluation of the patients on the time point of 1year after diagnosis were shown, as well as the treatments receivedduring the whole 1 year. Two patients were excluded from the analysisbecause one patient lacked the evaluation of 1 year, and the otherpatient did not receive any treatment until her disease was progressed.The disease progressive incidence of the patients who have receivedmultiple treatments of MASCT cell therapy is significantly reduced(p<0.0001), since only 1 patient (n=13, PD: 7.69%) had progressivedisease (Table 1). We also found that the average time to diseaseprogression was shorter (median: 6 months) of the patients received onlyconventional therapies, although average numbers of treatments receivedby each patient was relatively higher (11 months, Table 1 and FIG. 10A).Moreover, single MASCT cell therapy was not able to improve anyprogressive free outcome (data not shown) in this patient cohort,suggesting a causative effect for better clinical outcome attributed tomultiple treatments of MASCT cell therapies in patients with HCC, whichmay relate to the total amount of activated T cells infused.

TABLE 1 The comparison of RECIST evaluation between patients withhepatocellular carcinoma (B stage) with or without multiple treatmentsof MASCT cell therapy during 1 year after diagnosis Multiple MASCT NoMASCT n = 13 n = 15 p-values Overall response, No. (%) 2 (15.4) 1 (6.7)0.583^(a) Complete response (CR) 1 (7.7) 1 (6.7) 1^(a)    Partialresponse (PR) 1 (7.7) 0 (NA) 0.464^(a) Stable disease (SD), No. (%) 10(76.9) 1 (6.7) <0.0005^(b) Progressive disease (PD), 1 (7.69) 13 (86.7)<0.0001^(b) No. (%) Time to PD, median month 11 6 NT NA: not available;NT: not tested; ^(a)Analyzed by Fisher's Exact Test(2-sided);^(b)Analyzed by Pearson Chi-Square (2-sided)

Our study, for the first time, demonstrated that specific responses of Tcells against tumor antigens can be robustly raised in vivo, and suggestMASCT cell therapy as a safe and practical immunotherapy improving theimmunologic function and clinical outcome of patients with HCC.

Second Analysis of Clinical Benefits of MASCT

We have retrospectively studied the disease control rate (DCR) ofpatients with stage B HCC, who were diagnosed after 2012 and werecontinuously treated (with or without MASCT) and regularly followed-upfor longer than one year (FIG. 7B and Table 2). Through reviewing ofmedical records, we found seventeen patients had received conventionaltreatments (Group Con), such as resection, TACE, and RFA (radiofrequencyablation) (FIG. 8B). Meanwhile, fifteen patients had repeatedly receivedMASCT treatments (≥3) every 2-3 months, in addition to the conventionaltreatments (Group Con+MASCT, FIG. 9B). For these 15 patients, weperformed routine blood tests and blood biochemistry tests before andafter treatment. And no skin rashes, fatigue, fever, diarrhea, anemia,thrombocytopenia, or any other severe side effects were reported, whichindicates that MASCT is well tolerant. One year after diagnosis, the DCRwas 80% (12 out of 15) of Group Con+MASCT including 4 patient withoverall responses (1 patient with CR and 3 patients with PR), and 8patients with stable disease (SD). This DCR was significantly better(p<0.0001) than the DCR of Group Con (17.65%), in which only 3 out of 17patients shown as controlled disease (Table 2 and FIG. 9B). Moreover, wehave compared the median time to disease progression in the patients whohad been evaluated as PD in both group, and found that the time waslonger for the patients of Group Con+MASCT (11 months) than that ofGroup Con (6 months). The average number of conventional treatmentsreceived by each patient of Group Con+MASCT was 2.6 (FIG. 10B), ascompared to the average number for Group Con which was 3.71. These dataclearly showed that MASCT treatment brings up clinical benefits to stageB HCC patients.

TABLE 2 The comparison of disease control rate (DCR) between Stage B HCCpatients who had received conventional therapies combined with (GroupCon + MASCT) or without (Group Con) multiple treatment of MASCT celltherapy during 1 year after diagnosis. Con + MASCT Con (n = 15): (n =17): p-values Disease control rate (DCR), 12 (80%) 3 (17.65%)<0.0001^(b) No. (%) Overall response rate 4 (26.67%) 1 (5.88%) 0.161^(a)(ORR) Complete response (CR) 1 (6.67%) 1 (5.88%) 0.726^(a) Partialresponse (PR) 3 (20%) 0 0.092^(a) Stable disease (SD), No. 8 (53.33%) 2(11.76%) 0.021^(a) Progressive disease (PD), 3 (20%) 14 (82.35%)<0.0001^(b) No. (%) Time to PD, median month 11 6.5 ^(a)Analyzed byFisher's Exact Test (2-sided); ^(b)Analyzed by Pearson Chi-Square(2-sided)

Materials and Methods

Patients

The HCC patients from the department of liver diseases, Nanfang Hospitalof Southern Medical University who have received MASCT cell therapy mustsign the patients' informed consent before the treatment. All of thesepatients received infusions of the resulting T cells every 1-3 monthswith 5-10×10⁷ cells/kg body weight. The eligible criterion included: agebetween 25 and 80, an Eastern Cooperative Oncology Group (ECOG)performance status score of no more than 2, a life expectancy of morethan 3 months, and without severe cardiovascular disease, autoimmunedisease, or pregnancy.

Interim Analysis Study Design

The medical records of patients with HCC from a computerized database inthe Department of Liver Diseases, Nanfang Hospital of Southern MedicalUniversity were reviewed. This database recorded the clinical pathologicinformation of these patients included details about age, gender, tumorcharacteristics, BCLC stage, treatment, and outcome. 100 patients with Bstage of HCC were diagnosed in the department after 2012. Among them, 33patients were enrolled in this study since they were continuouslytreated in the Department for at least 1 year. Out of them, 3 patientswho received only once MASCT cell therapy were not further analyzed,since they did not follow the protocol of repeating treatments. The restpatients (n=30) were assigned into one of two treatment groups accordingto the patients' preference: patients in group 1 received standardtherapies only, while patients in group 2 received standard treatmentscombined with multiple MASCT cell therapy treatments. One patient ingroup 1 (n=13) was excluded since the patient lacked the evaluation of 1year. Another patient in group 2 (n=15) was excluded since she did notreceive any treatment until her disease was progressed. Thecharacteristics of the patients in these two groups are shown in FIG. 8Aand FIG. 9A. The primary endpoint was to investigate the rate ofpatients with progressive disease (PD) in one year and the time to PD.This study was approved by the Ethics Committee of Nanfang Hospital ofSouthern Medical University.

Safety Endpoint in Interim Analysis

For the 15 patients who received multiple MASCT cell therapy treatments,we have done the routine blood tests and the blood biochemistry testbefore and after MASCT cell therapy treatments respectively. Nosignificant difference was detected for the inflammation associatedindicators, such as leucocyte count, neutrophil count and lymphocytecount. No clear variety of liver function associated indicators wasdetected either, such as AST, ALT and total bilirubin. Moreover, no skinrash, fatigue, fever, diarrhea, anemia and thrombocytopenia wereobserved.

Second Retrospective Analysis

During the second retrospective analysis, the medical records ofpatients were used from a computerized database in the Centre of LiverDiseases, Nanfang Hospital of Southern Medical University. This databaserecorded the clinical pathologic information of all patients, includingage, gender, tumor characteristics, stages, treatment, and RECISTevaluation. We have analyzed the DCR of patients who were diagnosed asstage B HCC after 2012, and were continuously treated and regularlyfollowed-up in this center for at least one year (FIG. 7B). There werein total 53 patients matching the criteria. Out of them 17 patients hadtaken SOC but no MASCT, and were distributed into Group Con. The other36 patients with stage B HCC had received MASCT in addition toconventional treatments. However, 21 out of them were excluded sincethey had not finished the requirements of repeating treatments (≥3) ofMASCT in one year. The remaining 15 patients were distributed into GroupCon+MASCT. The characteristics of the patients in these two groups areshown in FIGS. 8B and 9B. According to Response Evaluation Criteria InSolid Tumors (RECIST v1.1), patients were determined as having acomplete response (CR), partial response (PR), stable disease (SD), orprogressive disease (PD) according to a computed tomography (CT) scan.The primary objective was to investigate the disease control rate ofstage B HCC patients one year after diagnosis. This study was approvedby the Ethics Committee of the Nanfang Hospital, Southern MedicalUniversity.

Cells Preparation

Peripheral blood mononuclear cells (PBMCs) from HCC patients wereobtained by density gradient centrifugation on Lymphoprep (NycomedPharma, Oslo, Norway). The adherent monocytes were continued to becultured in AIM-V medium with 1000 U/mL GM-CSF and 500 U/mL IL-4 todifferentiate into immature dendritic cells (DCs). The resultingimmature DCs were pulsed by multiple tumor antigens peptide pool (1μg/mL/peptide), followed up with TLR agonists, to differentiate intomature DCs. Meanwhile, the non-adherent PBMCs were maintained in AIM-Vmedium with anti-CD3 (eBioscience, San Diego, Calif.) and interleukin-2(rIL-2; R&D Systems, Minneapolis, Minn.), and were then co-cultured withmature DCs for 7-14 days (such as 7-10 days, or 9-13 days) in thepresence of cytokines. Patients received infusions of the resulting Tcells every 1-3 months with 5-10×10⁷ cells/kg body weight.

Immunofluorescence

Dendritic cells (DCs) were cultured in a Chamber slides (ThermoScientific, USA) and pulsed with FITC labeled peptides with or withoutliposome encapsulation. After 2 h, DCs were labeled with DAPI (MolecularProbes) and LYSOTRACKER™ (Molecular Probes) to identify nuclei andlysosomes, respectively. The fluorescent images were recorded using aconfocal laser scanning microscopy (TCS SP5II, Leica,Ernst-Leitz-Strasse, Germany).

Flow Cytometry

Antibodies for cell surface staining were obtained from BD Biosciences(anti-human CD3-PE, CD3-FITC, CD8-PerCP, CD8-APC, CD56-PE, NKG2D-APC,CD4-FITC, CD4-PerCP, CD107a-FITC, CD25-APC, CD45RO-FITC,CD27-PerCPCY5.5, CD57-APC, CCR7-PE, PD-1-PE). Antibodies for monocyteand dendritic cell surface staining were also obtained from BDBiosciences (anti-human CD14-APC, CD80-PE, CD83-APC, CD86-FITC,HLA-DR-FITC). Antibodies for intracellular cytokine staining wereobtained from BD Biosciences (anti-human IFN-γ-APC, TNF-α-PECY7,GranzymeB-FITC, FoxP3-PE). Intracellular cytokine staining was performedby fixing and permeabilizing cells with cytofix/cytoperm (BDBiosciences). Flow cytometry was performed using FACS Cantoll (BDBiosciences) flow cytometers and data was analyzed with the Flowjoprogram.

ELISA for Cytokine Detection

The supernatants of mature DCs or cultured T cells were centrifuged toremove particulate debris and stored at −80° C. until use. IL-12p70 andIL-10 were measured by specific ELISA kits (eBioscience) according tothe manufacturer's protocols. IFN-γ, TNF-α and IL-4 were detected byProcarta Plex Multiplex Immunoassays (Affymetrix).

Functional and Cytotoxicity Studies

HepG2 or Huh7 were cultured in DMEM supplemented with 10% inactivatedfetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin,Glutamax, MEM NEAA, (Gibico, Carlsbad, Calif.). The cytotoxicity assaywas performed as in the manufacturer's manual. HepG2 or Huh7 cells werewashed with D-PBS (Invitrogen) and co-cultured with the patient'seffector T cells at an effector:target (E:T) ratio of 40:1 in 96-wellround-bottom plates in triplicates in AIM-V for 4 hours. Cytotoxicitywas shown as the percentage of maximal LDH released after lysis andmeasured by the Cytotox 96 Assay kit (Promega G1780, Canada).

Proliferation Assay and IFNγ Production of Antigen Specific T Cells

PBMCs from patients were plated (1×10⁶ cells/well) in AIM-V mediumcontaining 50 U/mL IL-2 and stimulated with 10 μg/mL peptide pool for 3days. To determine the proliferative percentage of specific T cells,FACS analysis was performed as described in the Click-iT EdU Alexa Fluor488 Flow Cytometry Assay Kit (Invitrogen). The IFNγ production ofspecific T cells was also detected by intracellular cytokine stainingand FACS analysis. PBMCs incubated with 10 μg/mL irrelevant peptide wereused as negative controls. The results were shown as a fold indexcompute by specific peptide group: irrelevant peptide group.

ELISPOT Assay

PBMCs from patients were plated (1×10⁶ cells/well) in AIM-V mediumwithout any cytokines on cell culture plate, and were further stimulatedwith irrelevant peptides, MASCT antigen peptide pool, and individualantigen peptides respectively for 48 h. The PBMCs were then transferredonto a 96-well ELISPOT assay plate (U-CyTech Biosciences) for IFNγdetection. The PBMCs were further stimulated with peptides for another16 h. The ELISPOT assay was performed and analyzed according to themanufacturer's instructions. The number of spot-forming units wasdetermined with computer-assisted image analysis software (ChampSpot;Saizhi). The responses were shown as spot-forming units per 10⁵PBMC/well. Results were demonstrated as an IFNγ-producing fold indexcomputed by the ratio of specific peptide stimulation to irrelevantpeptide stimulation.

Discussion

Adoptive cell therapy (ACT) are recently well accepted as an effectivelyalternative therapy to treat cancer patients, especially for those whohad failed to be treated with conventional cancer therapies, such assurgery, radiation therapies, and chemotherapies. Though the tumorspecific TILs had successfully treated metastatic melanoma patients withmore than 50% overall responses [21], and the anti-CD19 CAR-modified Tcells had shown extraordinary clinical benefits both in chroniclymphatic leukemia (CLL) and acute lymphatic leukemia (ALL) [22, 23],the ACT using tumor specific T cells still faced to great challenge. Inthe tumor environment, tumor cells, which express and present aparticular tumor antigen on their surface, could be targeted by thetumor infiltrating T cells which specifically recognized the epitopes ofthe particular tumor antigen. After recognition, the T cells willrelease cytotoxic factors such as perforin and granzyme B, as well asthe functional cytokines such as IFNγ and TNFα to lysate the tumorcells. However, this mechanism of immune surveillance is high likely tobe shut down in cancer patients. The large number of inhibitor cellssuch as T_(REG), the lack of tumor specific TILs, and the loss of tumorantigen expression in tumor cells could all contribute to the failure.

Here, we have presented a novel and practical strategy, MASCT, bypreparing multiple tumor antigens activated T lymphocytes ex vivo frompatients' PBMCs. In MASCT strategy, we used long antigen peptidesinstead of tumor antigen protein or tumor lysate. We demonstrated thestimulation of T cells with multiple tumor antigen epitopes at the sametime, which may more effectively prevent the immune escape of tumorcells caused by the loss of a particular tumor antigen expression. Inour study, distinct patterns of specific T cell responses against eachkind of tumor antigens were observed in different HCC patients aftermultiple treatments of MASCT. This may be due to the diversity ofantigen expression and presentation, as well as the variability of thetumor microenvironment. This phenomenon also suggests the need to usemultiple antigens to target tumor cells instead of a single tumorantigen. Moreover, each tumor antigen peptide was designed to contain20-40 amino acids which enables the presentation of both class I andclass II HLA molecules on the DCs in patients with different kinds ofHLA subtypes. Indeed, we have observed specific immune responses againsttumor antigens in different HCC patients by both the T cellproliferation assay and the IFNγ stimulation assay, as well as thedecrease of T_(REG) in patients' PBMCs after multiple treatment ofMASCT. And these immune responses were gradually enhanced duringmultiple treatment of MASCT, indicating that repeating treatment maycorrelate with a better clinical outcome.

It has been reported that 240 million people worldwide (includingone-tenth population in China) are chronically infected with HBV, whohave high risk to develop HCC later in their life. With few effectiveconventional therapies, HCC in China is a kind of cancer with highmorbidity and mortality. Trying to solve this problem, we have appliedMASCT in HCC patients to stimulate specific T cell responses againsttumor antigens and HBV associated antigens, intending to improve theclinical outcome of patients by the combination treatment of MASCT andconventional therapy. Through retrospective analysis, we haveinvestigated the clinical effects of patients with stage B HCC afterreceiving multiple treatments of MASCT. The one-year DCR wassignificantly increased in the group of patients treated with MASCTevery 2-3 months combined with conventional therapies (Group Con+MASCT),compared to the control group (80% vs. 17.65%). We have further analyzedthe DCR two years after diagnosis of the 12 patients from GroupCon+MASCT who demonstrated disease control in the first year. Excludingtwo patients whose disease courses were less than two years, 9 out of 10patients still demonstrated as disease control (data not shown).

For the first time, our study has demonstrated that specific responsesof T cells against tumor antigens can be strongly induced and increasedin vivo by MASCT, and that MASCT treatment is a well tolerantimmunotherapy to improve both the immunologic function and diseasecontrol of patients with HCC. The same principle and methodology isundergoing a perspective and randomized clinical trial for HCC patients(NCT02026362), and is being explored for other tumors as well.Furthermore, we speculate that MASCT treatment can be combined withimmune checkpoint blockade therapy, such as anti-PD1 antibody, given thefact that anti-PD1 antibody therapy brings only clinical benefits toaverage 20% patients in different kinds of cancer. The 80%non-responding patients are thought to have not enough pre-existingtumor specific T cells, which may be able to be rescued by MASCTtreatment.

REFERENCES

-   1. A. J. Gehring et al., Gastroenterology 137, 682 (2009).-   2. E. Mizukoshi et al., Hepatology 43, 1284 (2006).-   3. V. R. Cicinnati et al., Int J Cancer 119, 2851 (2006).-   4. S. Idenoue et al., Clin Cancer Res 11, 1474 (2005).-   5. T. Ito et al., Hepatology 31, 1080 (2000).-   6. J. L. Chen et al., J Immunol 165, 948 (2000).-   7. J. L. Marshall et al., J Clin Oncol 23, 720 (2005).-   8. S. Walter et al., Nat Med 18, 1254 (2012).-   9. N. Nishida et al., Cancer Res 54, 3107 (1994).-   10. K. Schag et al., Clin Cancer Res 10, 3658 (2004).-   11. C. N. Boss et al., Clin Cancer Res 13, 3347 (2007).-   12. H. Suzuki et al., J Transl Med 11, 97 (2013).-   13. L. H. Butterfield et al., Clin Cancer Res 12, 2817 (2006).-   14. H. Komori et al., Clin Cancer Res 12, 2689 (2006).-   15. A. J. Gehring et al., J Hepatol 55, 103 (2011).-   16. J. Yuan et al., Proc Natl Acad Sci USA 105, 20410 (2008).-   17. B. Martin et al., J Hepatol (2014).-   18. A. Schurich et al., Hepatology 53, 1494 (2011).-   19. A. G. Chapuis et al., Proc Natl Acad Sci USA 109, 4592 (Mar. 20,    2012).-   20. D. J. Powell, Jr., M. E. Dudley, P. F. Robbins, S. A. Rosenberg,    Blood 105, 241 (2005).-   21. Restifo N P, Dudley M E, Rosenberg S A. Adoptive immunotherapy    for cancer: harnessing the T cell response. Nature reviews    Immunology 2012; 12:269-281.-   22. Porter D L, Levine B L, Kalos M, Bagg A, June C H. Chimeric    antigen receptor-modified T cells in chronic lymphoid leukemia. N    Engl J Med 2011; 365:725-733.-   23. Grupp S A, Kalos M, Barrett D, Aplenc R, Porter D L, Rheingold S    R, et al. Chimeric antigen receptor-modified T cells for acute    lymphoid leukemia. N Engl J Med 2013; 368:1509-1518.

Example 2—A Case Study of a Patient with Metastatic Cervical CancerTreated with MASCT and Cloning of Tumor Specific T Cell Receptors

Cervical cancer is the second most common gynecologic malignant tumor,and frequently occurs in patients with human papilloma virus (HPV)infection. Effective treatment for cervical cancer (including surgeryand concurrent chemoradiation) can yield cures in 80% of women withearly stage disease (stage I-II). However, vascular invasion, incompletelymphadenectomy are most common factors predicting poor prognosis inearly stage cervical cancer. Patients with vascular invasion confirmedby pathological specimens are more likely to develop metastatic diseasein the near future post-surgery. Here we present an immunotherapy namedMASCT (Multiple Antigen Stimulating Cellular Therapy), to treat a HPV+metastatic cervical cancer patient with multiple tumor antigen pulseddendritic cells (DCs) and T lymphocytes stimulated by these DCs.

Patient W J, female, was diagnosed with cervical cancer with vascularinvasion at age 41, and was tested positive with Human Papilloma Virus(HPV) DNA. She underwent curative resection, and a five-monthchemo-radio therapy. The patient took a second HPV DNA test, and wasconfirmed to be negative in serum HPV DNA.

About two years after the curative resection and chemo-radio therapy,the patient was diagnosed to have metastasis tumor on the rightsacroiliac joint bone according to Magnetic Resonance Imaging (MRI) andEmission Computed Tomography (ECT) (FIG. 13A). The patient then receivedten local radiotherapy treatments, followed by three MASCT treatment,administered one per month. The MASCT treatment used PBMCs from thepatient's own peripheral blood to prepare dendritic cells pulsed with apool of 18 antigen peptides, including a core group of 12tumor-associated antigen peptides, as well as a cervical cancer-specificgroup of 6 antigen peptides derived from viral proteins of HPV. Briefly,monocytes from the patient's PBMCs were differentiated into immature DCsand then pulsed with multiple synthetic peptide antigens includingtumor-associated antigens and HPV antigens. The semi-mature DCs werefurther stimulated by TLR ligands to differentiate into mature DCs(mDCs). Half of mDCs were subcutaneous injected to the patient.Maintaining T cells were prepared by culturing non-adherent PBMCs withanti-CD3 antibody (e.g., OKT3), and IL2. The other half of mDCs wasco-cultured with the maintaining T cells for another 7-9 days beforeinfusion. The patient was confirmed to have HLA-A2 serotype(HLA-A0201⁺).

After the three MASCT treatments, the patient's ECT results showed thatthe right sacroiliac joint bone metastasis was reduced, and no newmetastasis was detected (FIG. 13B), indicating positive treatmentoutcome of MASCT. The patient received four additional MASCT treatmentsadministered with an interval of about 1 month or 2 months. After atotal of 6 MASCT treatments, a sample of the patient's PBMC was obtainedand tested with an ELISPOT assay to determine whether the patient had atherapeutically effective MHC-restricted T cell response to the antigenpeptide pool and each of the antigen peptides within the pool. TheELISPOT results (FIG. 13E) demonstrated enhanced T-cell response to thecervical carcinoma antigen peptide pool, and individual antigen peptideswithin both the core group of tumor-specific antigen peptides (such ashTERT, p53, CEA, and RGS5), and the cervical cancer-specific group oftumor antigen peptides (such as HPV-3 and HPV-5). The patient's ECTafter a total of 7 MASCT showed further reduction of the rightsacroiliac joint bone metastasis, and no new metastasis sites (FIG.13C), indicating that the MASCT treatment regimen was successful inreducing tumor burden in the patient and in preventing tumor progressionand further metastasis.

Based on the patient's specific immune response, the antigen peptidepool was customized to provide a patient-specific antigen peptide poolby saving the responsive peptides that had induced specific responsesand removing the non-responsive peptides that did not induce specificresponses. The patient was further treated with 3 cycles of MASCTprepared using the patient-specific antigen peptide pool (referredherein as “precise MASCT”). After the three precise MASCT, The patient'sECT showed no development of the right sacroiliac joint bone metastasis,and no new metastasis sites (FIG. 13D). The patient was evaluated ashaving stable disease (SD). The patient-specific antigen peptide poolfurther boosted the specific responses as demonstrated by the ELISPOTassay (FIG. 13F). In particular, several hTERT peptides, p53-1 peptide,CEA peptides, and RGS5 peptide yielded the strongest specific response.We are in the process of cloning the CEA and telomerase specific TCRsfrom this patient.

The antigen peptide pool was further adjusted based on the specificimmune response of the patient, and the patient was treated with a2^(nd) precise MASCT using the further adjusted peptide antigen pool.

A summary of the patient's treatment history is shown in FIG. 14.

Our study provides MASCT as a safe treatment, which has reduced themetastasis of the cervical cancer patient. Tumor antigens specific Tcell responses could be robustly raised in cervical cancer patientsafter MASCT treatment, and were even further boosted afterpatient-specific antigens selection. Additionally, our study provides apromising method to clone tumor specific TCRs from cancer patients, whohave shown enhanced immunological responses, as well as clinicalbenefits after immunotherapy with patient-specific antigens.

Example 3—Brief Description of a Phase 1/2 MASCT Clinical Trial StudyProtocol

A Phase 1/2 MASCT clinical trial study, entitled “Multiple AntigenSpecific Cell Therapy (MASCT) for Hepatocellular Carcinoma (HCC)Patients After Radical Resection or Radio Frequency Ablation (RFA)”,commenced in July 2013, and is registered at the online databaseClinicalTrials.gov with identifier NCT02026362. Description of someaspects of the study can be found athttps://clinicaltrials.gov/ct2/show/NCT02026362, which is incorporatedherein by reference. This clinical study is ongoing.

The Phase 1/2 MASCT study is a 1:1 randomized, open-label, multi-centerstudy that aims to investigate the safety and efficacy of an embodimentof the MASCT method in treating hepatocellular carcinoma (HCC) patientsthat have previously received curative resection, such as resection orRFA, as anti-HCC therapy. The study is carried out in multiple sites inChina, including Nanfang Hospital of Southern Medical University(Guangzhou, PRC), Third Affiliated Hospital of Sun Yat-Sen University(Guangzhou, PRC), Cancer Center of Sun Yat-Sen University (Guangzhou,PRC), PLA 302 Hospital of China (Beijing, PRC), and Fujian CancerHospital (Fuzhou, PRC). The patients are randomly stratified into acontrol group and a test group (1:1 in number of patients of the twogroups) based on standard prognostic factors, such as age, sex, previoustreatment regimen, and clinical performance status. Patients in thecontrol group receive a standard of care (SOC) according to currentmedical practice, including liver-protection treatment and anti-viraltreatment against Hepatitis B virus (HBV) using nucleoside analoguedrugs. In China, HCC is highly associated with HBV infection. Patientsin the test group receive the same SOC treatment, plus MASCT treatment,which involves administration of dendritic cells loaded with a total of14 antigens, including hTERT, surviving, p53, and CEA, and activated Tcells induced by the dendritic cells. The MASCT treatment is repeatedevery three weeks for a total of three times. Each patient in the twostudy groups will receive treatment for about 9 weeks unless the patientexperiences disease progression or unacceptable toxicity. If patientshave not progressed after 9 weeks, treatment may be continued at theinvestigator's discretion. Patients will be followed for about 2.5 yearsor until death or disease progression of all patients, whichever occursearlier.

Approximately 100 patients are planned to be enrolled, treated andevaluated in the study. Patients must fulfill all of the followingcriteria to be eligible for admission to the study.

-   -   1. The patient is diagnosed as hepatocellular carcinoma(HCC);    -   2. The patient underwent radical operation of HCC within 8 weeks        before enrollment;    -   3. The number of tumors is no more than 2    -   4. No cancer embolus in the main portal vein, first branch of        hepatic duct, first branch of hepatic vein, or inferior vena        cava;    -   5. No portal lymph node metastasis    -   6. No extra-hepatic metastasis;    -   7. Complete tumor resection without residual tumor at the        surgical margins as confirmed by enhanced CT or MRI imaging        within 4 week (including 4 weeks) after radical operation;    -   8. If an increased serum AFP level was detected of the patient        before the radical operation, the AFP level should be returned        to normal within 8 weeks;    -   9. Child-Pugh Score ≤9;    -   10. ECOG Performance status (ECOG-PS)≤2;    -   11. The expected survival time is more than 2 years;    -   12. Tests of blood, liver and kidney meeting the following        criteria        -   a. WBC>3×10⁹/L        -   b. Neutrophil counts >1.5×10⁹/L        -   c. Hemoglobin ≥85 g/L        -   d. Platelet counts≥50×10⁹/L        -   e. PT is normal or The extend time <3s        -   f. BUN≤1.5 times the upper-limit,        -   g. Serum creatinine≤1.5 times of the upper-limit    -   13. Patient consent obtained and signed according to local        Institutional and/or University Human Experimentation Committee        requirements and/or a central Institutional Review Board (IRB)        or other as appropriate.

Patients who fulfill any of the following criteria are not eligible foradmission to the study:

-   -   1. Women who are pregnant or during breast feeding or plan to be        pregnant within 2 years;    -   2. Extra-hepatic metastasis or liver residual tumor;    -   3. Cancer embolus in the main portal vein, first branch of        hepatic duct, first branch of hepatic vein, or inferior vena        cava;    -   4. 6 months before enrollment: the duration of systemic and        continuous use of immunomodulatory agents (such as interferon,        thymosin, traditional Chinese medicine) was longer than 3        months;    -   5. 6 months before enrollment: the duration of systemic and        continuous use of the immunosuppressive drugs (such as        corticosteroids drug) was longer than 1 month;    -   6. Received any cell therapy (including NK, CIK, DC, CTL, stem        cells therapy) within 6 months prior to enrollment;    -   7. Positive for HIV antibody or HCV antibody;    -   8. Have a history of immunodeficiency disease or autoimmune        diseases (such as rheumatoid arthritis, Buerger's disease,        multiple sclerosis or diabetes type 1);    -   9. Patients who suffered from other malignant tumor within 5        years before enrollment (except skin cancer, localized prostate        cancer or cervix carcinoma);    -   10. Patients with organ failure;    -   11. Patients with serious mental disease;    -   12. Drug addiction within 1 year before enrollment, including        alcoholism;    -   13. Participated in other clinical trials within 3 months before        screening;    -   14. Other reasons the researchers deem unsuitable for the study.

The primary objective of the study is to demonstrate that MASCT plus SOCtreatment is superior to foundation treatment alone with respect to (1)number of patients having tumor recurrence or metastasis within 2 yearsas a measure of efficacy; (2) time from operation to tumor recurrence ormetastasis within 2 years as a measure of efficacy; (3) number ofpatients having adverse events within 2 years as a measure of safety andtolerability. The secondary objective of the study is to compare MASCTplus foundation treatment to foundation treatment alone with respect totheir effects on (1) HBV markers, including HBeAg; (2) serum HBV DNAload; and (3) patients' quality of life. Additional clinical endpoints,such as overall response rate (RR), complete response (CR), partialresponse (PR) and stable disease rate (SDR) of the two patient groupswill be compared. Tumor response and progression will be assessed usingRECIST criteria (v1.1). Safety will be assessed on the basis of vitalsigns, clinical laboratory findings, and adverse events graded accordingto the NCI CTCAE version 4.02, 15 Oct. 2009.

Other clinical trials investigating the clinical efficacy and toxicityof embodiments of the MASCT method, similar to the Phase 1/2 MASCTclinical trial in HCC, will be conducted to treat patients sufferingfrom liver cancer, lung cancer, colon cancer, cervical cancer, lymphoma,renal carcinoma, breast cancer, pancreatic cancer, gastric cancer,esophageal cancer, ovarian cancer, and brain cancer.

Example 4—T Cell Activation Protocols

This example compares the in vitro specificity and function of cytotoxicT cells prepared using various exemplary T cell activation protocols,including different duration and cycles of T cell co-culture, and thepresence or absence of an immune checkpoint inhibitor, such as anti-PD-1monoclonal antibodies, during the co-culture.

Use of Anti-PD-1 Antibody

FIG. 15 shows a schematic of the experimental setup for preparingactivated T cells. Peripheral blood mononuclear cells (PBMCs) from HCCpatients that were positive in HBV were obtained by density gradientcentrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway) on Day 0.The adherent monocytes were continued to be cultured in AIM-V mediumwith 1000 U/mL GM-CSF and 500 U/mL IL-4 to differentiate into immaturedendritic cells (DCs). The immature DCs were pulsed by HBV core antigenpeptide (5 μg/mL) as well as TLR agonists to differentiate into matureDCs. A fraction of PBMCs were also pulsed with the same antigen peptide,and fixed. Meanwhile, the non-adherent PBMCs were maintained in AIM-Vmedium with anti-CD3 (eBioscience, San Diego, Calif.) and interleukin-2(rIL-2; R&D Systems, Minneapolis, Minn.) until Day 8 to obtainmaintaining T cells. The maintaining T cells were then co-cultured withmature DCs alone, or in combination with an anti-PD-1 antibody(nivolumab, or SHR-1210) or a negative control IgG4 from Day 9 to Day 13in the presence of cytokines to provide activated T cells, (i.e.,cytotoxic T lymphocytes or CTL). On Day 14, the fixed, peptide-pulsedPBMCs were added to the CTLs and co-cultured for 5 days for a secondcycle of T cell stimulation. Alternatively, a second batch ofpeptide-pulsed mature DCs can be used instead of the fixed,peptide-pulsed PBMCs for the second cycle of T cell stimulation.

Peptide-pulsed mature DCs and T cells from Day 8 were analyzed usingFACS to quantify subpopulations that expressed PD-L1 or PD-1respectively. Activated T cell samples from the co-cultures wereobtained on Day 13 and Day 18, and assayed by staining with pentamersfollowed by FACS analysis. Staining with pentamers and other multimers(such as dextramers) indicates presence of TCRs on the activated T cellsspecifically recognizing MHC-peptide complexes, thereby providing ameasure of specificity of the activated T cells. The activated T cellsfrom Day 13 and Day 18 were also stimulated with the tumor antigenpeptides pool, and IFNγ production by the activated T cells wasdetermined by intracellular cytokine staining followed by FACS analysis.As IFNγ was produced by T cells specifically activated by the tumorantigen peptides, the IFNγ production assay provides a measure of thecytotoxic function of the peptide-specific T cells.

As shown in FIG. 16A, about 89.1% of peptide-pulsed mature DCs expressedPD-L1. FIG. 16B shows a significant increase in the percentage of PD1⁺ Tcells among all CD3⁺ T cells in the PBMCs from four independent donorsby Day 8.

When anti-PD1 antibodies were introduced to the co-culture of T cellsand DCs, both specificity and function of the activated T cells in vitroincreased significantly (FIGS. 17A-17D) as compared to activated T cellsprepared without anti-PD-1 antibodies or with a negative control IgG4.In particular, SHR-1210 (Hengrui Medicine) enhanced the in vitro IFNγproduction by the activated T cells as compared to nivolumab (FIG. 17D).

Duration and Number of Stimulation

FIG. 18 shows a schematic of the experimental setup for preparingactivated T cells. Peripheral blood mononuclear cells (PBMCs) fromhealthy donors that were positive in EBV were obtained by densitygradient centrifugation on Lymphoprep (Nycomed Pharma, Oslo, Norway) onDay 0. The adherent monocytes were continued to be cultured in AIM-Vmedium with 1000 U/mL GM-CSF and 500 U/mL IL-4 to differentiate intoimmature dendritic cells (DCs). The immature DCs were pulsed by multipletumor antigens peptide pool (1 μg/mL/peptide) as well as TLR agonists todifferentiate into mature DCs. A fraction of PBMCs were also pulsed withthe same tumor antigens peptide pool, and fixed. Meanwhile, thenon-adherent PBMCs were maintained in AIM-V medium with IL2, IL7, IL15and IL21 to obtain maintaining T cells until Day 8. The maintaining Tcells were then co-cultured with mature DCs alone, or in combinationwith an anti-PD-1 antibody (nivolumab, or SHR-1210), or a negativecontrol IgG4 from Day 9 to Day 18 in the presence of cytokines toprovide activated T cells, (i.e., cytotoxic T lymphocytes or CTL). OnDay 14, a fraction of the activated T cells were mixed with the fixed,peptide-pulsed PBMCs, and cultured for 5 days for a second cycle of Tcell simulation. Alternatively, a second batch of peptide-pulsed matureDCs can be used instead of the fixed, peptide-pulsed PBMCs for thesecond cycle of T cell stimulation.

Activated T cell samples from the co-cultures were obtained on Day 13and Day 18, and assayed by staining with pentamers or dextramersfollowed by FACS analysis. The activated T cells from Day 13 and Day 18were also stimulated with the tumor antigen peptides pool, and IFNγproduction by the activated T cells was determined by intracellularcytokine staining followed by FACS analysis.

As shown in FIG. 19A, a higher percentage of peptide-specific T cellswere present in the co-culture after 10 days of stimulation as comparedto 5 days of stimulation. Among all culturing conditions tested, 10 daysof co-culture with one time stimulation in the presence of anti-PD1antibodies resulted in the highest specificity and cytotoxic function ofthe CD8⁺ T cells, as measured by multimer staining (FIG. 19B), and IFNγproduction (FIG. 19C).

Total number of T cells in samples taken from Day 8, Day 10, Day 13, andDay 15 of the 1-time stimulated co-culture were quantified for twopreparations using PBMCs from two different donors. As shown in FIGS.20A-20B, among all samples tested, the highest number of total T cellswas found in the co-culture taken on Day 15 in the presence of SHR-1210anti-PD1 antibody.

Additionally, PD-1 surface expression was quantified in non-adherentPBMC cells treated with an anti-PD1 antibody (nivolumab or SHR-1210) ornegative control IgG4, and PMA on Day 0. PBMCs treated anti-PD1antibodies showed reduced PD-1 expression level on the cell surface,which would be expected if the anti-PD1 antibodies could internalize thesurface PD1 (FIGS. 21A-21B).

In summary of the above in vitro characterizations of T cells activatedusing the various preparation protocols, use of anti-PD1 monoclonalantibodies in the PBMC culture improved the specificity and function ofthe activated cytotoxic T cells. Anti-PD1 monoclonal antibodies seemedto both enhance the general proliferation of T cells in vitro, andpromote internalization of PD1 molecules that normally express on thesurface of T cells. Compared to nivolumab, SHR-1210 anti-PD1 antibodyyielded activated T cells with higher specificity and function. Otherimmune checkpoint inhibitors, such as anti-PD-L1 antibodies oranti-CTLA-4 antibodies, can be used in place of the anti-PD1 antibodiesfor preparing activated T cells with enhanced specificity and cytotoxicfunction.

Example 5—Cancer Precision Immunotherapy Clinical Practice Case Analysis

This example describes a study aimed at predicting the response rate andeffectiveness of Major Histocompatibility Complex (MHC) Class Irestricted immunotherapy such as PD-I inhibitor or multiple antigenspecific cancer therapy (MASCT) by evaluating the cancer cell drivermutation and human leukocyte antigen (HLA) class I gene mutation loadusing the next generation sequencing (NGS).

By the next generation sequencing (NGS) of 333 cancer associated genesand using Dirichlet Multinomial Mixture package in RStudio software forclassification, 35 cancer samples were clustered into two subgroups.Mutation load of HLA-I genes was evaluated from each sample, andneoantigens were predicted from non-synonymous point mutations. Thecombined mutational information was used to predict the response of 35cancer patients to MHC-I restricted immunotherapy. Among the 35 patientswhose samples have been sequenced and analyzed, five patients receivedPD-1 inhibitor monotherapy, MASCT monotherapy or combination therapy.Two patients, who had high HLA-I gene mutation load (predicted asnon-responders), received PD-1 inhibitor (KEYTRUDA®) and MASCTcombination therapy or MASCT monotherapy for more than three times.These two patients were clinical evaluated as having progressive disease(PD). Three patients, who had low HLA-I gene mutation load and moreneoantigens (predicted as responders), received PD-1 inhibitor(KEYTRUDA®) and MASCT combination therapy or MASCT monotherapy for morethan four times. Two of the three patients were clinical evaluated ashaving partial response (PR), and one patient was evaluated as havingstable disease (SD).

The NGS analysis of mutation in cancer associated genes described inthis example successfully predicted the clinical response to MHC-Irestricted immunotherapy among patients who received MASCT monotherapyor MASCT combination therapy with PD-1 inhibitor. The prediction methodcan enable improved efficiency and precision immunotherapy forindividual cancer patients.

Introduction

Next Generation Sequencing (NGS) can sequence a tumor tissue in a rapidand high-throughput manner to provide a large amount of mutation data ofthe tumor tissue. Bioinformatics methods can be applied to the mutationdata to obtain clinically significant mutations, thereby providinguseful information for the cancer patient in the following areas: 1.clinical and molecular stratification; 2. selection of suitable drugtargets; and 3. prediction of effectiveness of an MHC-I restrictedimmunotherapy. Such information can provide precision intervention foreach patient, and bring cancer immunotherapy to an era of precisionmedicine. Cancer precision immunotherapy, such as precision immune celltherapy, precision immunological drug therapy, promise to become animportant breakthrough in cancer precision therapy, greatly improvingpatients' quality of life, and increasing patient's survival time (see,for example, Qian Qijun, Mengchao Wu. Precision cancer immunotherapy:From theory to practice [J]. Chin J Cancer Biother, 2015,22(2):151-158).

Materials and Methods

Sample Source

Tumor samples from 35 patients were sequenced. Among the patients, therewere 18 males, 17 females, age 27-88, with an average age of 56. Tumorsamples were obtained from the patients and used for sequencing analysisfocusing on 333 OncoGxOne™ cancer-associated genes and the HLA-I genes.Next-generation sequencing (NGS) was performed to obtain geneticmutation information in tumor tissue, such as point mutation, indel,fusion, copy number variation, etc., with a focus on 333cancer-associated genes. Five of the 35 patients were further treatedwith PD-1 inhibitor (Keytruda) monotherapy, MASCT monotherapy, or thecombined therapy (FIG. 22). Informed consent was obtained from patientsfor all clinical trials.

HLA-I Subtyping

Low quality reads and adaptor sequences were removed from the rawsequencing data, then Polysolver analysis tool (see, for example, ShuklaS A, Rooney M S, Raj asagi M, et al. Comprehensive analysis ofcancer-associated somatic mutations in class I HLA genes. NatureBiotechnology, 2015, 33:1152-1158) was used for HLA-I subtypingprediction.

Neoantigen Prediction

Based on HLA-I subtyping results of each patient, multiple algorithmsincluding NetMHC 3.4 were employed to analyze amino acid sequences ofthe point mutation loci of the 333 cancer-associated genes from thetumor tissue. The affinity of the mutated amino acids to thecorresponding HLA-I molecules of the patient was predicted, the affinitydifference between wild-type and mutant antigen peptides was compared,and mutant antigen peptides with higher affinity than that of wild-typewere selected. The T-cell receptor (TCR) binding affinity was thenpredicted using the mutant antigen peptides selected above with highaffinity to HLA-I molecules, and the TCR binding affinity differencebetween wild-type and mutant antigen peptides was compared. The mutantantigen peptides with higher binding affinity to TCR than that ofwild-type antigens were selected as predicted neoantigens that caninduce immune response. The sequences of these predicted neoantigenswere mapped to the entire human genome of healthy individuals, andneoantigens with potential cross-reacting sequences were removed fromthe pool, in order to avoid adverse effects in clinical trials.

Statistics

1.1 The open source software RStudio version 0.99.473 was used toperform analysis on the number of point mutation loci, number of geneswith point mutation, number of indel loci, number of genes with indel,number of fusion genes, number of genes with copy number variation, andthe total numbers of mutated loci and genes. These mutageneticcharacteristics were employed for stratified analysis of 35 tumorsamples with the Dirichlet Multinomial Mixture (DMM) model (see, forexample, Holmes I K, Quince C. Harris. Dirichlet Multinomial Mixtures:Generative Models for Microbial Metagenomics [J]. PLoS ONE, 2012,7(2):e30126).

1.2 The “pheatmap” package was used to generate clustering plot for the35 tumor tissue samples based on mutation load of each of the 333cancer-associated genes. The characteristic information of each tumorsample was added to the cluster plot, such as clinical trial groupinginformation, grouping information of whether there was DNAmismatch-repair (MMR) deficiency or not, and DMM grouping information.

1.3 The statistical difference of the number of HLA-I gene mutations ineach DMM group based on the RStudio software was tested usingMann-Whitney rank-sum test, with p<0.05 as statistically significant.

Results

Stratified Cluster Analysis of 333 Cancer-Associated Genes

Mutagenetic characteristics data were statistically analyzed for 333cancer-associated genes in each tumor sample, including number of pointmutation loci, number of genes with point mutation, number of indelloci, number of genes with indel, number of fusion genes, number ofgenes with copy number variation, and the total numbers of mutated lociand genes, and these data were used for DMM stratified analysis on 35tumor tissue samples. As depicted in FIG. 23A, the best cluster of the35 samples is two groups. The labeled clustering of each sample is shownin FIG. 23B, which depicts the effective separation of members of thetwo groups based on Minimum Distance Separation (MDS1) distance: 14tumor samples were clustered into group DMM 1, and 21 tumor samples wereclustered into group DMM 0.

Heatmap was generated for 35 tumor samples based on number of pointmutations, number of indel loci, and number of loci with copy numbervariations detected for each of the 333 cancer-associated genes in eachtumor sample, and cluster analysis was done for the samples and genes.As shown in FIG. 24A, two major clustering branches were observed forthe 35 tumor samples. After adding clinical or molecular label for eachsample, the cluster result was found to be inconsistent with that ofcancer clinical types: tumor samples of the same cancer clinical typehave different mutagenetic spectra, while some tumor samples ofdifferent cancer clinical types share similar mutations. The differencebetween cancer molecular classification and clinical types observed inthis study is consistent with other reports (see, for example, Golub TR, Slonim D K, Tamayo P, et al. Molecular classification of cancer:class discovery and class prediction by gene expression monitoring [J].Science, 1999, 286(5439):531-537). The clustering based on MMRdeficiency type is also inconsistent with the clustering result shown inthe heatmap. However, it was noted that the DMM groups had similarclassification with the clustering results, only one sample showedinconsistency between DMM classification and clustering (FIG. 24A).

By measuring the total mutation number at HLA-A, HLA-B and HLA-C locidetected in each tumor sample, and comparing to the tumor tissueclustering result in the heatmap, a significant difference of HLA-I genemutation load between the two DMM groups was observed (FIG. 24B).

Percentage of HLA-I Gene Mutation in the Total Mutation Number

Further analysis of HLA-I gene mutation number indicated statisticallysignificant difference between the two DMM groups. The HLA-I genemutation load of the 14 tumor samples in DMM group 1 (red) issignificantly higher than that of the 21 tumor samples in DMM group 0(green), with a p-value of 1.076e-5 (FIG. 25A). Moreover, the ratio ofHLA-I gene mutation load to the total mutation load of DMM group 1 issignificantly higher than that of DMM group 0 (FIG. 25A), suggestingthat HLA-I gene mutation may be a critical internal mutation of cancercells of DMM group 1. High mutation load of the HLA-I gene mayfacilitate escape of cancer cells from immune surveillance, leading topossible ineffectiveness of MHC-I restricted immunotherapy.

Five of the 35 cancer patients received PD-1 inhibitor (KEYTRUDA®)monotherapy, MASCT monotherapy, or their combined therapy (FIGS. 22,25B). Two patients with high mutation load on the HLA-I genes (DMMgroup 1) were predicted to be unresponsive to MHC-I restrictedimmunotherapy. After treated with the combination therapy of PD-1inhibitor (KEYTRUDA®) and MASCT or MASCT monotherapy for at least 3cycles, these two patients were clinically evaluated as havingprogressive disease (PD) or ineffective clinical benefit response (CBR),as predicted. Three patients with low mutation load on the HLA-I genes(DMM group 0) were predicted to be responsive to MHC-I restrictedimmunotherapy. Two of these three patients were treated with PD-1inhibitor (Keytruda) monotherapy or (Keytruda) and MASCT combinationtherapy for 5 cycles, and were clinically evaluated as having partialresponse (PR) or obvious CBR; one of the three patients was treated withMASCT for 4 cycles, and was clinically evaluated as having stabledisease, as predicted.

Typical Patient Case Analysis

Patient ID 1-LQL

Patient ID 1-LQL had lung adenocarcinoma, and was predicted to be of DMMgroup 1 based on clustering analysis. 55 mutations were detected atHLA-I gene loci, and was classified as HLA-I high mutation load (FIG.22). MHC-I restricted immunotherapy was predicted to be clinicallyineffective.

After receiving 3 cycles of PD-1 inhibitor (KEYTRUDA®) treatment and 4cycles of MASCT treatment, the patient experienced respiratory failureand died due to ineffective disease control and pleural effusion. Thispatient was clinically evaluated as having progressive disease (PD), aspredicted by clustering analysis.

Patient ID 2-SJS

Patient ID 2-SJS had esophageal cancer, and was predicted to be of DMMgroup 1 based on clustering analysis. 8 mutations were detected at HLA-Igene loci, and was classified as HLA-I high mutation load (FIG. 22),suggesting ineffectiveness with MHC-I restricted immunotherapy.

After receiving 3 cycles of MASCT monotherapy, this patient wasclinically evaluated as having progressive disease (PD), as predicted byclustering analysis.

Patient ID 3-HJL

Patient ID 3-HJL, female, age 73, had transitional cell carcinoma ofleft renal pelvis, with multiple metastases towards left adrenal gland,left supraclavicular and mediastinal lymph nodes and two lungs. Thispatient was clustered into DMM group 0 based on Next GenerationSequencing (NGS) results, with 2 mutations detected at HLA-I gene loci,and was classified as HLA-I low mutation load. 6 neoantigens werepredicted based on HLA-I subtyping of the patient. Therefore, MHC-Irestricted immunotherapy was predicted to render effective CBR for thispatient.

This patient was diagnosed to have transitional cell carcinoma of leftrenal pelvis and received radical surgery. Metastasis at left adrenalgland was discovered 2 years later, therefore, radiofrequency ablation(RFA) was applied. PET-CT scan after the RFA indicated multiplemetastases at left supraclavicular and mediastinal lymph nodes and twolungs, with the biggest tumor size of ˜2 cm in diameter, so the patientstarted to receive chemotherapy. After the fourth chemotherapy, CTre-examination indicated ineffective cancer control by chemotherapy(FIGS. 26A-C). The patient was then treated with PD-1 inhibitor(KEYTRUDA®) and MASCT combination therapy. After 3 cycles of combinationtherapy, the tumor size was found to have shrunk for ˜50% by CT scan(FIG. 26D). After 5 cycles of PD-1 inhibitor (KEYTRUDA®) and MASCTcombination therapy, CT scan suggested disappearance of tumor in lungs,stable disease at mediastinal lymph nodes, and disappearance of swollenleft supraclavicular lymph nodes. The disease condition was clinicallyevaluated as partial response (PR, FIG. 26E). The clinical result was aspredicted.

Patient ID 4-LKS

Patient ID 4-LKS, male, age 61, experienced stage IV moderatelydifferentiated left lung adenocarcinoma after radical surgery, togetherwith metastases at brain and mediastinal lymph nodes. This patient wasclustered into DMM group 0 based on NGS results, with 2 mutationsdetected at HLA-I loci, and was classified as HLA-I low mutation load. 6neoantigens were predicted based on HLA-I subtyping of the patient.Therefore, MHC-I restricted immunotherapy was predicted to rendereffective CBR for this patient.

This patient received radical surgery of the left lung tumor 13 yearsago, followed by 4 cycles of adjuvant chemotherapy and mediastinal CTscans. Intracranial metastases were detected during the re-examination,with the tumor size of ˜3 cm (FIG. 27A), accompanied with righthemiplegia. After 20 cycles of targeted radiation (dose informationunavailable), the tumor shrank in size (FIG. 27B). The patient thenreceived PD-1 inhibitor (KEYTRUDA®) monotherapy. After 2 cycles oftreatment, the CT re-examination indicated further shrinkage of theintracranial tumor (FIG. 27C), and clinical symptom showed gradualrecovery of muscle strength in the hemiparesis side. After receiving 6cycles of PD-1 inhibitor (KEYTRUDA®) monotherapy, CT re-examinationsuggested further improved intracranial tumor and edema status (FIG.27D), and clinical symptom indicated gradual recovery of muscle strengthin the hemiparesis side and the ability of performing fine movement.After finishing radiation therapy and 6 months of PD-1 inhibitor(KEYTRUDA®) monotherapy, the patient was clinically evaluated as havingpartial response (PR). This clinical result was as predicated by oursequencing analysis.

Discussion

In patients with “reversible HLA-I deficiencies,” after adoptive T cellimmunotherapy, T cells can locally secrete IFN-γ and upregulate theexpression of HLA-I molecules. Therefore, it will be of greater clinicalsignificance to assess whether patients have “irreversible HLA-Ideficiencies” prior to receiving adoptive T cell immunotherapy.

The sequencing results based on the five tumor tissue samples discussedabove suggested weaker correlation between CBR after receiving PD-1inhibitor (KEYTRUDA®) and/or MASCT and DNA MMR deficiency status(mutations detected at MLH1, MSH2, MSH6 and PMS2 loci), compared to thatbetween CBR and HLA-I gene mutation load. MMR deficiency may lead toelevated mutation rate in cancer cells, which in turn may result in moreneoantigens, theoretically. However, if these cancer cells have low orno expression of HLA-I molecules, the neoantigens would not beeffectively presented for recognition and to trigger cytotoxic effect byimmune cells. Therefore, the availability of HLA-I molecules foreffective neoantigen presentation in cancer cells is one of therequirements for MHC-I restricted immunotherapy to be effective. Thepatient case 1.LQL supported this hypothesis: although many mutationswere detected in the tumor tissue (a total of 3243 mutations detected),since HLA-I gene mutation load was also quite high, PD-1 inhibitor(KEYTRUDA®) therapy or MASCT did not have significant clinical effect.

Since HLA-I genes have high polymorphism, after obtaining the HLA-I genemutation information in tumor tissue, further testing is necessary todetermine whether and to what extent the amino acid mutation at certainlocus would affect antigen presentation by HLA-I molecules. In thisstudy, statistical analysis was carried out to perform stratifiedanalysis on cancer patients, and cluster them into one responsive groupand one non-responsive group to MHC-I restricted immunotherapy, based onHLA-I gene mutation load in the tumor tissue of each patient, so that acorrelation between clinical response and HLA-I gene mutation load canbe measured. According to the 5 case studies, 3 patients with low HLA-Imutation load showed effective CBR to MHC-I restricted immunotherapy,while 2 patients with high HLA-I mutation load did not show effectiveCBR to such therapy. This example reports the first clinical discoverythat bioinformatics analysis on NGS data of patient samples can beperformed to provide prognosis of cancer immunotherapy (such as usingPD-1 inhibitor KEYTRUDA®) and adoptive T cell therapy (such as MASCT)based on number of neoantigens and HLA-I gene mutation load, therebyproviding patients with precision immunotherapy. Clinical studies with alarger sample size are underway to corroborate this discovery.

Example 6—a Case Study of a Patient with Colorectal Cancer Treated withNeoantigens-MASCT

Patient XMZ, male, age 58, was diagnosed with colon cancer and receivedcolectomy. Pathological analysis indicated Stage II colon cancer. Threeyears after the colectomy, the patient received CTC monitoring, whichshowed a CTC amount of 194 cells/10 mL. The patient received threetreatment cycles of precision MASCT as outlined in FIG. 28.

Briefly, as the first step, a tumor biopsy sample from the patient wassequenced and analyzed using the ONCOGXONE™ cancer-associated genes plusHLA panel (Admera Health). The ONCOGXONE™ plus HLA panel used IlluminaMiSeq or HiSeq sequencing platform to sequence about 150-400 genes thatare specific to the cancer type, including HLA loci. Agilent SURESELECT™target enrichment kit was used to enrich the target sequences in thesample. Target sequence regions spanned about 2-5.4 Mb of each genelocus, covering all exons, UTRs and relevant intron regions. Averagecoverage depth was about 100 times. Genomic variations, including pointmutations, indels, rearrangements, and copy number variations (CNV)based on the sequencing data were determined.

ONCOGXONE™ plus HLA analysis of the patient's sample revealed that thepatient had a G12A point mutation in the KRAS gene, which may reducesensitivity of the patient to treatment with monoclonal antibodiesagainst EGFR, such as cetuximab, or panitumumab. The patient had a pointmutation at residue Q399 of the XRCC1 gene, suggesting a potentialenhanced response to platinum-based chemotherapeutic drugs. The patienthad mutations in MTHFR and TYMS, suggesting a potential enhancedresponse to 5-FU. The patient had mutations in CYP2D6, suggesting apotential reduced response to tamoxifen or opioid analgesics.Potentially beneficial drugs predicted based on the sequencing resultsinclude anti-PD-1 antibodies (such as OPDIVO® or KEYTRUDA®), PD-0332991(Palbociclib), and trametinib (such as MEKINIST®).

Additionally, HLA subtyping and mutation analysis based on thesequencing results revealed that the patient had two deletion mutationin the HLA-I genes, including one in the HLA-A locus, and one in theHLA-C locus. HLA-I subtyping and mutation load results of the patient'stumor sample are shown in Tables 3 and 4 below. Functional analysis ofthe patient's HLA loci suggested that MHC-I restricted therapy, such asT cell-based therapy, might be effective for the patient.

TABLE 3 HLA-I subtyping results Locus A1 A2 A 0206 2402 B 1502 5101 C0302 0801

TABLE 4 Mutations in HLA-I class genes Gene Chromosome Location WTMutation Frequency Type Seq. depth HLA-A chr6 29912028 AG A 0.1783deletion 129 HLA-C chr6 31236715 CG C 0.9429 deletion 245

Based on the sequencing analysis, four candidate neoantigens werediscovered (FIG. 29A). Two new antigen peptides were designed based onneoantigens MTHR-A222V and MLL3-C988F respectively. Also, a neoantigenpeptide KRAS_G12V from our antigen peptide library was chosen. Together,16 antigen peptides (including hTERT, p53, Survivin, NY-ESO-1, MET,MUC1, Kras-3, neoantigen 1+2) were included in the antigen peptide poolfor the MASCT treatments that were specific to the patient's sigmoidcolon tumor.

After three cycles of precision MASCT treatments using the antigenpeptide pool comprising the neoantigen peptides, the number ofcirculating tumor cells (CTC) in the patient was reduced (FIG. 29B).PBMC samples from the patient were assayed by ELISPOT to assessantigen-specific T cell response. The ELISPOT results (FIG. 29C)revealed that the patient's PBMC had T lymphocytes exhibiting strongspecific response against MUC1 and the neoantigens (neoantigen 1+1,Kras-3), while specific response against hTERT, p53, surviving,NY-ESO-1, and MET was also significant. Specific T cells against tumorneoantigens and tumor associated antigens were present in the patient'sPBMC, and proliferated over the course of the MASCT treatments. Patientreported improved vigor and physical strength, as well as changes inphysical appearance (such as hair and beard turning black) afterreceiving the three cycles of MASCT treatment.

Example 7—Prognosis and Stratification of Patients for MASCT Treatment

This example presents an exemplary model for providing prognosis andstratification of patients receiving MHC-restricted therapy, such asMASCT treatments (including Precision MASCT), based on the number ofneoantigens (i.e. neoantigen load) and mutation load of HLA molecules inthe patients.

The mutation load of HLA molecules and neoantigens in tumor samples from40 cancer patients were determined and analyzed using the methodsdescribed in Example 5. Patients were predicted to benefit from theMHC-restricted therapy if the patients had: (1) no mutation in B2M; (2)no mutation in functional regions (such as leader peptide sequence, a1domain, a2 domain, or a3 domain) of HLA genes; (3) less than 2 mutationsin HLA-I A, B, or C genes; and (4) more than 5 neoantigens. Patientswere predicted to potentially benefit from the MHC-restricted therapy ifthe patients had: (1) no mutation in B2M; (2) no mutation in functionalregions (such as leader peptide sequence, a1 domain, a2 domain, or a3domain) of HLA genes; (3) at least 2 mutations and less than 10mutations in HLA-I A, B, or C genes; and (4) less than 5 neoantigens.Patients were predicted to have no benefit from the MHC-restrictedtherapy if the patients have: (1) one or more mutations in B2M; or (2)at least 10 mutations in HLA-I A, B, or C genes. 9 patients receivedMHC-restricted therapy, including MASCT and/or immune checkpointblockade treatment. Table 5 below shows the clinical response of thepatients in the three prognosis groups.

TABLE 5 Prognosis of patients for MHC-restricted therapy Potential NoPrognosis groups Benefit benefit benefit Number (total = 40) 17 (42.5%)21 (52.5%) 2 (5%) Receive MHC-restricted therapy 5 3 1 Respond toMHC-restricted 4 2 0 therapy No response to MHC-restricted 1 1 1 therapyResponse rate 80% 66%  0% Success rate of prognosis 80% NA 100%

Example 8—Safety of MASCT in Patients with Hepatocellular Carcinoma

45 patients with hepatocellular carcinoma were treated with MASCT, andtheir clinical data, including clinical response, liver and renalfunction, routine blood examination results, and adverse reactions, werecollected and retrospectively analyzed. The patients did not receive anyother immunotherapy, and their latest treatments (surgery, radiotherapy,chemotherapy) before receiving MASCT were completed at least one monthor more prior to the MASCT. FIG. 30A shows the clinical characteristicsof the 45 patients.

Cells were prepared for the MASCT according to the method described inExample 1. Briefly, PBMCs were isolated from each patient on Day 1, andthe adherent cells were induced into DCs. DCs were loaded with 14 kindsof multiple antigen peptides to prepared the mature DCs (mDCs). A smallportion of mDCs were injected to the patients subcutaneously on Day 8.The non-adherent cells from the PBMC sample were co-cultured with therest of mDCs from Day 7, and induced into cytotoxic T cells (CTLs),which were infused intravenously on Day 26. The mDCs and CTLs werecharacterized to ensure quality control. In the mDCs, the percentage ofCD80⁺ cells was 98.5±5%, the percentage of CD83⁺ cells was 88±10%, thepercentage of CD86⁺ cells was 98.4±3%, and the percentage of HLA-DR⁺cells was 98.8±2%. The mDCs secreted high level of IL-12 (985±312 μg/mL)and low level of IL-10 (53±10 μg/mL). In the CTLs, the percentage ofCD3⁺CD8⁺ cells was 83±10%, and the percentage of CD3⁺CD56⁺ cells was24±5%. The CTLs secreted high level of IFN-γ (1222±650 μg/mL), and lowlevel of IL-10 (6.8±5.0 μg/mL).

All 45 patients had improved clinical conditions with varying degreesafter receiving the MASCT treatment. Most patients reported improvedvigor, appetite, sleep, and physical strength. The main adverse eventsafter infusion of the active immunocytes were moderate fever (2 cases,4.44%). The fever occurred after about 1 hour of the CTL infusion, andin both cases, the fever was no more than 38.5° C. The patients resumednormal body temperature after rest, drinking water and physical cooling.No other severe adverse events were observed.

FIG. 30B shows the results of routine blood examination of the 45patients before MASCT treatments and after the last MASCT treatments.There was no abnormality observed in the routine blood examinationresults of any patient. The White Blood Cell (WBC) number (P=0.0411),and neutrophil (Neu) numbers (P=0.0015) showed significant changesbefore and after the MASCT treatments, but are within normal ranges, andthus do not lead to significant clinical effects. Hemoglobin (HB) andplatelet (PLT) numbers showed no statistically significant change.Statistical analysis was performed using t test using SPSS 19.0software.

FIG. 30C shows the ALT, AST, TBIL, CR and BUN levels in the 45 patientsbefore MASCT treatments and after the last MASCT treatments. There wasno abnormality observed in the liver or renal function of any patient.Data from 2 patients before MASCT treatments was unavailable. AST(p=0.0198) and TBIL (p=0.0177) levels showed statistically significantchange after the MASCT treatment, and ALT also had a tendency toincrease, which have clinical significance. CR and BUN levels had nostatistical significant change after the MASCT treatments.

After 4-6 MASCT treatments, liver function data were available for 6visits (baseline level to after 5 MASCT treatments). FIG. 30D shows thechange in the ALT and AST levels in 10 patients over the course of 5MASCT treatments. 2 patients with large fluctuations in the levels wereexcluded, including one patient that received liver transplantation atthe 6^(th) visit, which resulted in an increased level of ALT to 288IU/L; and a second patient that received TACE therapy, which resulted inan increased level of 572 IU/L at the 6^(th) visit. Comparing the ALTand AST levels in the patients over time, there was no statisticallysignificant change.

The results demonstrate that MASCT treatment is safe for HCC patients.

What is claimed is:
 1. A method of preparing a population of activated Tcells, the method comprising: a) Inducing differentiation of apopulation of monocytes into a population of dendritic cells; b)Contacting the population of dendritic cells with a plurality of tumorantigen peptides to obtain a population of dendritic cells loaded withthe plurality of tumor antigen peptides, wherein the plurality of tumorantigen peptides comprises at least one epitope having an amino acidsequence selected from the group consisting of SEQ ID NOs: 36-38 and41-44; and c) Co-culturing the population of dendritic cells loaded withthe plurality of tumor antigen peptides and a population of non-adherentperipheral blood mononuclear cells (PBMCs) to obtain the population ofactivated T cells; wherein the population of monocytes and thepopulation of non-adherent PBMCs are obtained from a population of PBMCsfrom an individual.
 2. The method of claim 1, wherein step b) comprisescontacting the population of dendritic cells with the plurality of tumorantigen peptides in the presence of a composition that facilitates theuptake of the plurality of tumor antigen peptides by the dendriticcells.
 3. The method of claim 1, wherein step b) further comprisescontacting the population of dendritic cells loaded with the pluralityof tumor antigen peptides with a plurality of Toll-like Receptor (TLR)agonists to induce maturation of the population of dendritic cellsloaded with the plurality of tumor antigen peptides.
 4. The method ofclaim 1, wherein step c) further comprises contacting the population ofactivated T cells with a plurality of cytokines to induce proliferationand differentiation of the population of activated T cells.
 5. Themethod of claim 1, wherein the population of non-adherent PBMCs iscontacted with an immune checkpoint inhibitor prior to the co-culturing,or wherein step c) comprises co-culturing the population of dendriticcells loaded with the plurality of tumor antigen peptides and thepopulation of non-adherent PBMCs in the presence of an immune checkpointinhibitor.
 6. The method of claim 1, wherein the plurality of tumorantigen peptides comprises a first core group of general tumor antigenpeptides.
 7. The method of claim 6, wherein the plurality of tumorantigen peptides further comprises a second group of cancer-typespecific antigen peptides.
 8. The method of claim 6, wherein the firstcore group comprises about 10 to about 20 general tumor antigenpeptides.
 9. The method of claim 1, wherein the plurality of tumorantigen peptides comprises a neoantigen peptide.
 10. The method of claim1, wherein the plurality of tumor antigen peptides comprises at least 10tumor antigen peptides each comprising one or more epitopes encoded by acancer-associated gene selected from the group consisting of hTERT, p53,Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc,HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.
 11. The method of claim 1,wherein the plurality of tumor antigen peptides comprises at least 14tumor antigen peptides, wherein the at least 14 tumor antigen peptidescomprise epitopes of SEQ ID NOs: 1-35.
 12. The method of claim 1,wherein the concentration of each tumor antigen peptide in the pluralityof tumor antigen peptides is about 0.1-200 μg/mL.